Peptide agonists of glp-1 activity

ABSTRACT

Novel peptide agonists of GLP-1 activity useful for lowering blood glucose levels. The novel peptides comprise variants of the GLP-1 or the exendin-4 polypeptide sequence and are pharmacologically active and stable. These peptides are useful in the treatment of diseases that benefit from regulation of excess levels of blood glucose and/or regulation of gastric emptying, such as diabetes and eating disorders.

FIELD OF THE INVENTION

The present invention relates to novel peptide agonists of GLP-1activity. More specifically the invention relates to novel peptides thatlower blood glucose levels comprising variants of the exendin-4polypeptide sequence and peptide conjugates comprising variants of theGLP-1 or the exendin-4 polypeptide sequences which are pharmacologicallyactive and stable, and as agonists of GLP-1 activity are useful in thetreatment of diseases that benefit from regulation of excess levels ofblood glucose and/or regulation of gastric emptying, such as diabetesand eating disorders. The present invention also relates to methods ofpreparing said novel peptides, a composition, e.g., a pharmaceuticalcomposition, comprising a peptide of the invention and a physiologicallyacceptable carrier, to said peptide for use in therapy, a method oftreating a disorder and to the use of said peptide for the manufactureof a pharmaceutical composition for use in therapy.

BACKGROUND OF THE INVENTION

A number of hormones that lower blood glucose levels are released fromthe gastrointestinal mucosa in response to the presence and absorptionof nutrients in the gut. These include gastrin, secretin,glucose-dependent insulinotropic polypeptide (GIP) and glucagon-likepeptide-1 (GLP-1). The most potent substance known is GLP-1 (Ørskov,1992, Diabetologia 35:701-711). Glucagon-like peptide 1 (GLP-1) is aproduct of proglucagon, a 180 amino acid peptide (Drucker, 1998,Diabetes 47:159-169). The overall sequence of proglucagon contains the29-amino acid sequence of glucagon, the 36 or 37 amino acid sequence ofGLP-1 and the 34 amino acid sequence of glucagon-like peptide-2 (GLP-2),an intestinotrophic peptide. GLP-1 has a number of functions. It is aphysiological hormone that enhances the effect on insulin secretion innormal humans and is therefore an incretin hormone. In addition, GLP-1also lowers glucagon concentrations, slows gastric emptying, stimulates(pro)insulin biosynthesis, and enhances insulin sensitivity (Nauck,1997, Horm. Metab. Res. 47:1253-1258). The peptide also enhances theability for the β-cells to sense and respond to glucose in subjects withimpaired glucose tolerance (Byrne, 1998, Eur. J. Clin. Invest.28:72-78). The insulinotropic effect of GLP-1 in humans increases therate of glucose disappearance partly because of increased insulin levelsand partly because of enhanced insulin sensitivity (D'Alessio, 1994,Eur. J. Clin. Invest. 28:72-78). This has placed GLP-1 as a promisingagent for treatment for type II diabetes. Active fragments of GLP-1 havebeen found to be GLP-1(7-36) (SEQ ID NO: 114) and GLP-1(7-37) (SEQ IDNO: 124).

However, a major pharmacological problem with native GLP-1 is its shorthalf-life. In humans and rats, GLP-1 is rapidly degraded by dipeptidylpeptidase-1V (DPP-IV) into GLP-1(9-36)amide (SEQ ID NO: 125), acting asan endogenous GLP-1 receptor antagonist (Deacon, 1998, Diabetologia41:271-278). Several strategies circumventing this problem have beenproposed, some using inhibitors of DPP-IV and others DPP-IV resistantanalogues of GLP-1(7-36)amide (SEQ ID NO: 114) (Deacon, 1998,Diabetologia 41:271-278; Deacon et al., 1998, Diabetes 47:764-769;Ritzel, 1998, J. Endocrinol. 159:93-102; U.S. Pat. No. 5,545,618;Pederson, 1998, Diabetes 47:1253-1258).

Exendins, another group of peptides that lower blood glucose levels havesome sequence similarity (53%) to GLP-1[7-36]NH₂ (SEQ ID NO: 114) (Gokeet al., 1993, J. Biol. Chem. 268:19650-55). The exendins are found inthe venom of Helodermatidae or beaded lizards (Raufman, 1996, Reg.Peptides 61:1-18). Exendin-3 is present in the venom of Helodermahorridum, the Mexican beaded lizard and exendin-4 is present in thevenom of Heloderma suspectum, the Gila monster. Exendin-4 differs fromexendin-3 at just positions two and three. The cDNA encoding theexendin-4 precursor protein, a 47 amino acid peptide fused to the aminoterminus of exendin-4 has been cloned and sequenced (Pohl et al., 1998,J. Biol. Chem. 273:9778-9784 and WO98/35033). Both exendin-3 andexendin-4 stimulate an increase in cellular cAMP production in guineapig pancreatic acinar cells by interacting with exendin receptors(Raufman, 1996, Reg. Peptides 61:1-18). Exendin-3 causes a biphasicincrease in cellular cAMP production, but a monophasic increase inamylase release in pancreatic acinar cells. In contrast, exendin-4causes a monophasic increase in cAMP production and does not alteramylase release.

Exendin-4 is a strong GLP-1 receptor agonist on isolated rat insulinomacells (Goke et al., 1993, J. Biol. Chem. 268:19650-55). This is expectedas the (His Ala) domain of GLP-1 recognised by DPP-IV is not present inexendin-4 (Goke et al., 1993, J. Biol. Chem. 268:19650-55). Binding of[¹²⁵I]GLP-1 to the nucleus of the solitary tract was inhibitedconcentration-dependently by unlabelled GLP-1 and [Tyr39]exendin-4 (SEQID NO: 126) with Ki values of 3.5 and 9.4 nM respectively, and similarvalues are found in cell lines (Goke et al., 1995, Eur. J. Neurosci.7:2294-2300 and Goke et al., 1993, J. Biol. Chem. 268:19650-55).Further, exendin-4 given systemically lowers blood glucose levels by 40%in diabetic db/db mice (WO99/07404). Recently, Grieg et al. (1999,Diabetologia 42:45-50) has shown a long lasting blood glucose loweringeffect of once daily intraperitoneal injection of exendin-4 to diabeticob/ob mice). U.S. Pat. No. 5,424,286 discloses that a considerableportion of the N-terminal sequence is essential in order to preserveinsulinotropic activity (exendin-4(1-31) (SEQ ID NO: 127) andY³¹-exendin-4(1-31)) (SEQ ID NO: 148) whereas an N-terminally truncatedexendin (exendin-4(9-39) (SEQ ID NO: 128) has inhibitory properties.

The use of exendin-3, exendin-4 and exendin agonists has been proposedfor the treatment of diabetes mellitus, reducing gastric motility anddelaying gastric emptying and the prevention of hyperglycemia (U.S. Pat.No. 5,424,286, WO98/05351) as well as for the reduction of food intake(WO98/30231). There has been proposed ways of obtaining novel compoundsby modifying the native exendin sequences. One way is to attachlipophilic substituents to the molecule, e.g. as described in WO99/43708 which discloses derivatives of exendin with just one lipophilicsubstituent attached to the C-terminal amino acid residue.

A major approach has been to devise exendin analogues characterised byamino acid substitutions and/or C-terminal truncation of the nativeexendin-4 sequence. This approach is represented by the compounds ofWO99/07404, WO 99/25727 and WO 99/25728. WO99/07404 discloses exendinagonists having a general formula I that defines a peptide sequence of39 amino acid residues with Gly Thr in positions 4-5, Ser Lys Gln inpositions 11-13, Glu Glu Glu Ala Val Arg Leu in positions 15-21, Leu LysAsn Gly Gly in positions 26-30, Ser Ser Gly Ala in positions 32-35, andwherein the remaining positions may be occupied by wild-type exendinamino acid residues or may be occupied by specified amino acidsubstitutions. The formula I does not cover any exendin agonists oranalogues having specific amino acid deletions and/or being conjugatesas described herein, such as the novel compoundsdesPro³⁶-exendin-4(1-39) (SEQ ID NO: 101), exendin-4(1-39)-K₆ (SEQ IDNO: 92) or desPro³⁶-exendin-4(1-39)-K₆ (SEQ ID NO: 93).

WO 99/25727 discloses exendin agonists having a general formula I thatdefines a peptide sequence of from 28 to 38 amino acid residues with Glyin position 4 and Ala in position 18, and wherein the remainingpositions may be occupied by wild-type exendin amino acid residues ormay be occupied by specified amino acid substitutions. Formula I doesnot comprise a peptide sequence having Ser as the C-terminal amino acidand exendin agonists or analogues having specific amino acid deletionsand/or being conjugates as described herein, such as the novel compoundsdesPro³⁶-exendin-4(1-39) (SEQ ID NO: 101), exendin-4(1-39)-K₆ (SEQ IDNO: 92) or desPro³⁶-exendin-4(1-39)-K₆ (SEQ ID NO: 93). Further, formulaII of WO 99/25727 defines a peptide sequence similar to formula I, butincluding exendin derivatives having a C(1-10)alkanoyl orcycloalkylalkanoyl substituent on lysine in position 27 or 28.

When treating inappropriate post-prandial blood glucose levels thecompounds are administered frequently, for example one, two or threetimes a day.

WO 99/25728 discloses exendin agonists having a general formula I thatdefines a peptide sequence of from 28 to 39 amino acid residues withfixed Ala in position 18, and wherein the remaining positions may beoccupied by wild-type exendin amino acid residues or may be occupied byspecified amino acid substitutions. Said exendin agonists all correspondto a truncated exendin analogue having a varying degree of amino acidsubstitutions. Peptide sequences of from 34 to 38 amino acid residues donot have Ser C-terminally. A peptide sequence of 39 amino acid residuesmay have either Ser or Tyr C-terminally, but no further residues.Exendin agonists or analogues having specific amino acid deletionsand/or being conjugates according to the invention described herein arenot comprised by formula I. Further, formula II defines a peptidesequence similar to formula I, but including exendin derivatives havinga C(1-10)alkanoyl or cycloalkylalkanoyl substituent on lysine inposition 27 or 28.

WO 99/46283 (published 16.09.99) discloses peptide conjugates comprisinga pharmacologically active peptide X and a stabilising peptide sequenceZ of 4-20 amino acid residues covalently bound to X, where saidconjugates are characterised in having an increased half-life comparedto the half-life of X. X may be exendin-4 or exendin-3.

OBJECTIVE OF THE INVENTION

There is a need for compounds that lower blood glucose levels inmammals, and are stable and effective. Therefore, it is an objective ofthe invention to provide novel compounds that lower blood glucose levelsin mammals. Ideally, these should be effective when administered orally.It is a further object of the invention to provide novel peptideagonists of GLP-1 activity and/or exendin-4 activity. It is a stillfurther purpose of the invention to provide peptide agonists of GLP-1activity and/or exendin-4 activity having an increased half-life and/ora decreased clearance.

SUMMARY OF THE INVENTION

The invention is directed to a peptide conjugate comprising a peptide Xselected from the group consisting of

(a) an exendin having at least 90% homology to exendin-4;

(b) a variant of said exendin wherein said variant comprises amodification selected from the group consisting of between one and fivedeletions at positions 34-39 and contains a Lys at position 40 having alipophilic substituent; or

(c) GLP-1 (7-36) (SEQ ID NO: 114) or GLP-1 (7-37) (SEQ ID NO: 124)having at least one modification selected from the group consisting of:

-   -   (i) substitution of D-alanine, glycine or alpha-amino isobutyric        acid for alanine at position 8 and    -   (ii) a lipophilic substituent,        and Z, a peptide sequence of 4-20 amino acid units covalently        bound to said variant, wherein each amino acid unit in said        peptide sequence, Z is selected from the group consisting of        Ala, Leu, Ser, Thr, Tyr, Asn, Gln, Asp, Glu, Lys, Arg, His, Met,        Orn, and amino acid units of the general formula I

—NH—C(R¹)(R²)—C(═O)—  (I)

wherein R¹ and R² are selected from the group consisting of hydrogen,C₁₋₆-alkyl, phenyl, and phenyl-methyl, wherein C₁₋₆-alkyl is optionallysubstituted with from one to three substituents selected from halogen,hydroxy, amino, cyano, nitro, sulfono, and carboxy, and phenyl andphenyl-methyl is optionally substituted with from one to threesubstituents selected from C₁₋₆-alkyl, C₂₋₆-alkenyl, halogen, hydroxy,amino, cyano, nitro, sulfono, and carboxy, or R¹ and R² together withthe carbon atom to which they are bound form a cyclopentyl, cyclohexyl,or cycloheptyl ring, e.g. 2,4-diaminobutanoic acid and2,3-diaminopropanoic acid, with the proviso that X is not exendin-4 orexendin-3.

The peptide X is further characterised in being effective in improvingglucose tolerance in a diabetic mammal.

Furthermore, the invention is directed to a novel variant of a parentexendin, wherein said parent exendin has an amino acid sequence havingat least an 90% homology to exendin-4 and wherein said variant lowersthe blood glucose level in a mammal, binds to a GLP-1 receptor and hasat least one modification selected from the group consisting of (a)between one and five deletions at positions 34-38, and (b) contains aLys at position 40 having a lipophilic substituent attached to theepsilon amino group of said lysine.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the effect of Compound 1 (SEQ ID NO:101) (desPro³⁶-exendin-4(1-39)-NH₂) on blood glucose levels of mice, cf. Example25.

FIG. 2 shows the effect of Compound 2 (SEQ ID NO:93) (desPro³⁶-exendin-4(1-39)-Lys₆-NH₂ on the blood glucose levels of mice, cf.Example 25.

FIG. 3 shows the effect of Compound 5 (SEQ ID NO:89) (Gly⁸,Lys³⁷(palmitoyl)-GLP1-(7-36)(Human)-(Lys)₇-NH₂ on the blood glucoselevels of mice, cf. Example 25.

FIG. 4 shows in vivo degradation kinetics in rabbits after i.v.injection of 1 μmol/kg of Compound 4 and Compound (iii), cf. Example 27.

FIG. 5 is a plot of AUC (area under the curve) values (mean±SEM) forCompounds 2, 14-16, 18 and 19 in an oral glucose tolerance test (OGTT),cf. Example 28.

FIG. 6 shows a synthetic cDNA constructed for heterolog expression ofCompound 2 in yeast. The new construct was designated pYES0010, cf.Example 20.

FIG. 7 is a plot of dose-response on GTT in db/db mice based on relativeAUC_(0-240 min) values (mean±SEM) for Compound 2 and Compound (i), cf.Example 29.

FIG. 8 shows the effects of a maximal dose of Compound 2, i.e. 100nmol/kg i.p., on the oral glucose tolerance test (OGTT) whenadministered up to 24 hours before the OGTT.

DETAILED DESCRIPTION OF THE INVENTION

The compounds of the present invention include hitherto unknown deletionvariants of a parent exendin. In contrast to known substitution and/ortruncation variants of exendin-4(1-39) the novel compounds are believedto exhibit a stabilised alpha-helix structure with superior stabilityproperties and unreduced or enhanced binding properties. Moreover,conjugation of the novel variants, modified GLP-1(7-36)-NH₂ (SEQ ID NO:114), and modified GLP-1(7-37) (SEQ ID NO: 124) to specific shortpeptide sequences (Z) render stability to these compounds withoutcompromising the pharmacological properties. These conjugations conferin vivo stability and hydrophilicity to the peptide molecule. The Z iscomposed of amino-acid residues, and has alone no structuralcharacteristics in terms of α-helix conformation. However, from studiesusing both circular dichroism and nuclear magnetic resonance (NMR)spectroscopy, addition of Z dramatically alters the structuralcharacteristics of some peptides as evidenced by the increased amount ofα-helix conformation in the peptide. For example, circular dichroismdemonstrated that a Z-modified (Gly⁸)-GLP-1 (SEQ ID NO: 87) had muchmore α-helix conformation than (Gly⁸)-GLP-1 (SEQ ID NO: 87). Togetherwith the pharmacological results, the structural analyses suggest that Zis modifying the conformation of the peptide leading to higherenzyme-stability, but without losing its potency. Also the physical andchemical properties of peptides may be altered considerably byZ-modification with resulting impact on pharmacological formulationstrategy.

Exendin Variants

The exendin variant of the present invention is a variant of a parentexendin peptide having at least about 90% homology and most preferablyat least about 95% to exendin-4, which have exendin activity, e.g.,lowers the blood glucose level in a mammal and binds to a GLP-1receptor. In a preferred embodiment, the parent exendin peptide has anamino acid sequence which differs by five amino acids, preferably byfour amino acids, more preferably by three amino acids, even morepreferably by two amino acids, and still more preferably by one aminoacid residue from the amino acid sequence of exendin-4(1-39) (SEQ ID NO:102).

In one embodiment, the exendin variant comprises between one and fivedeletions at positions 34-38. Preferably the variant comprises between 1and 4 deletions at positions 34-38, more preferably between 1 and 3deletions at positions 36-38. Preferably the parent exendin isexendin-4, and a preferred variant included as peptide X in the peptideconjugates herein has an amino acid sequence wherein 1, 2 or 3 of thePro residues in positions 36, 37 and 38 have been deleted from the aminoacid sequence of exendin-4 and preferably from the amino acid sequenceof exendin-4(1-39) (SEQ ID NO: 102).

Coupling of a Z sequence to the X peptide herein is believed to increasethe stability of these compounds. Proline is a rigid amino acid that mayinterfere with the effect of Z to stabilise the structure of the Xpeptide. Deletion of one, two or all of the proline amino acids inpositions 36, 37 and 38 of the exendin backbone is therefore preferredin the peptide conjugates comprising a variant of a parent exendinaccording to the invention, as long as the efficacy of said conjugatesas measured in, e.g. an oral glucose tolerance test (OGTT) in diabeticdb/db mice, is not negatively affected.

In another embodiment, the variant comprises an additional residue atposition 40, a lysine residue which comprises a lipophilic substituentbound to the epsilon amino group of lysine via an amide bond. Thelipophilic substituent may be the acyl group of a straight-chain orbranched fatty acid or a straight-chain or branched alkaneα,ω-dicarboxylic acid. The acyl group may have the formulaCH₃(CH₂)_(n)CO—, wherein n is an integer from 4-38 and preferably from4-24. In a specific embodiment, the acyl group is selected from thegroup consisting of CH₃(CH₂)₆CO—, CH₃(CH₂)₈CO—, CH₃(CH₂)₁₀CO—,CH₃(CH₂)₁₂CO—, CH₃(CH₂)₁₄CO—, CH₃(CH₂)₁₆CO—, CH₃(CH₂)₁₈CO—,CH₃(CH₂)₂₀CO—, ad CH₃(CH₂)₂₂CO—. The acyl group may have the formulaHOOC(CH₂)_(m)CO—, wherein n is an integer from 4-38 and preferably from4-24. In a specific embodiment, the acyl group is selected from thegroup consisting of HOOC(CH₂)₁₄CO—, HOOC(CH₂)₁₆CO—, HOOC(CH₂)₁₈CO—,HOOC(CH₂)₂₀CO— and HOOC(CH₂)₂₂CO—. In a more specific embodiment, thelipophilic substituent is selected from the group consisting oftetradecanoyl, ω-carboxynonadecanoyl, 7-deoxycholoyl, choloyl, palmitoyland lithocholyl. In a most specific embodiment, the lipophilicsubstituent is palmitoyl.

Alternatively, the liphophilic substituent may have an NH group.Specific embodiments include but are not limited to the formulaeCH₃(CH₂)_(a)((CH₂)_(b)COOH)CHNHCO(CH₂)₂CO— wherein a and b are integersand a+b is an integer of from 8 to 33, preferably from 12 to 28;

CH₃(CH₂)_(c)CONHCH(COOH) (CH₂)₂CO — wherein c is an integer of from 10to 24;

CH₃(CH₂)_(d)CONHCH(CH₂)₂(COOH)CO— wherein d is an integer of from 8 to24;

COOH(CH₂)_(e)CO— wherein e is an integer of from 8 to 24;

—NHCH(COOH)(CH₂)₄NHCO(CH₂)_(f)CH₃ wherein f is an integer of from 8 to18;

—NHCH(COOH)(CH₂)₄NHCOCH(CH₂)₂COOH)NHCO(CH₂)_(g)CH₃ wherein g is aninteger of from 10 to 16; and—NHCH(COOH)(CH₂)₄NHCO(CH₂)₂CH(COOH)NHCO(CH₂)_(h)CH₃ wherein h is aninteger of 0 or from 1 to 22 and preferably from 10 to 16.

The exendin variants having a lysine residue at position 40 carrying alipophilic substituent optionally further comprise between one and fivedeletions, preferably between one and three deletions, at positions 34to 39, preferably at positions 34-38, such as [des Ser³⁹,Lys⁴⁰(palmitoyl)]exendin -4(1-39) (SEQ ID NO: 107), [des Pro³⁶,Lys⁴⁰(palmitoyl)]exendin-4(1-39) (SEQ ID NO: 110) and [des Pro³⁶,Lys⁴⁰(palmitoyl)]exendin-4(1-40) (SEQ ID NO: 152).

The variant may be in a most specific embodiment selected from the groupconsisting of:

Compound 1: des Pro³⁶-exendin-4(1-39)-NH₂ (SEQ ID NO:101),

des Pro³⁶-exendin-4(1-40)-NH₂ (SEQ ID NO: 139),

Compound 14: des Pro³⁶, Pro³⁷, Pro³⁸-exendin-4(1-39)-NH₂ (SEQ ID NO:132),

des Pro³⁶, Pro³⁷, Pro³⁸-exendin-4(1-40)-NH₂ (SEQ ID NO: 140),

des Pro³⁶, Pro³⁷-exendin-4(1-39)-NH₂ (SEQ ID NO: 130),

des Ala³⁵-exendin-4(1-39)-NH₂ (SEQ ID NO:105),

des Gly³⁴-exendin-4(1-39)-NH₂ (SEQ ID NO:106),

des Ser³⁹-(Lys⁴⁰(palmitoyl))exendin-4(1-39)-NH₂ (SEQ ID NO:107),

des Gly³⁴-(Lys⁴⁰(palmitoyl))exendin-4(1-39)-NH₂ (SEQ ID NO:108),

des Ala³⁵-(Lys⁴⁰(palmitoyl))exendin-4(1-39)-NH₂ (SEQ ID NO:109),

des Pro³⁶-(Lys⁴⁰(palmitoyl))exendin-4(1-39)-NH₂ (SEQ ID NO:110), and thefree acid thereof and a pharmaceutically acceptable salt thereof.

Modified GLP-1

A preferred modified GLP-1 included as peptide X in the peptideconjugates herein has an amino acid sequence of GLP-1 (7-36)-NH₂ (SEQ IDNO: 114) or GLP-1 (7-37) (SEQ ID NO: 124) having a substitution ofglycine for alanine at position 8. Alternatively, a preferred modifiedGLP-1 has an amino acid sequence of GLP-1 (7-36) (SEQ ID NO: 114) orGLP-1 (7-37) (SEQ ID NO: 124) having a substitution of glycine foralanine at position 8 and a lipophilic substituent, preferablypalmitoyl, on one lysine residue at position 26, 34 or 37. Thelipophilic substituent is preferably attached to the epsilon amino groupof said lysine and includes the specific embodiments described above forthe exendin variants. The modified GLP-1(7-36) (SEQ ID NO: 114) orGLP-1(7-37) (SEQ ID NO: 124) used as X in the conjugates of theinvention may be those cited in WO 99/43707 and WO 98/08871 comprising alipophilic substituent or, more preferably those GLP-1 analogues havinga glycine substitution at position 8. Preferred peptides X are

Gly⁸-GLP-1(7-36) (SEQ ID NO: 87),

Gly⁸-GLP-1(7-37) (SEQ ID NO: 123), and

Gly⁸-GLP-1(7-36)-Lys³⁷(palmitoyl) (SEQ ID NO: 147).

The compounds of the invention having a lipophilic substituent wouldhave a more protracted profile of action than the parent peptides asdemonstrated for GLP-1 derivatives in WO 98/08871.

Peptide Conjugates

The peptide sequence Z may be bound to the C-terminal or the N-terminalof the peptide sequence, X, or two peptide sequences may be boundindividually to both the C- and N-terminal of X. In case the nativepeptide X possesses a free C-terminal carboxylic acid, the peptidesequence Z may be attached to either the C-terminal of the peptide X orto the N-terminal of the peptide X, or the C- and N-terminal of X mayboth be bound to each individual peptide sequence Z. Alternatively, Zmay be bound to the nitrogen atom on the side chain of lysine, histidineor arginine or a carbonyl function on the side chain of glutamic acid oraspartic acid anywhere within the peptide sequence X. In one embodiment,Z may be attached to X within the sequence and to the N- and/ofC-terminal of X. Whether the sequence should be attached to the peptidesequence X at its C-terminal, at its N-terminal, or both, or within thepeptide sequence X depends on the specific peptide X and can be easilydetermined by the person skilled in the art. Preferably, X is bound to Zvia a peptide bond and preferably at the C-terminal of X.

One aspect of the invention is directed to a peptide conjugatecomprising a peptide X which reduces the blood glucose level in amammal, wherein X is (a) an exendin having at least 90% homology toexendin-4; (b) a variant of said exendin wherein said variant comprisesa modification selected from the group consisting between One and fivedeletions at positions 34-39 and contains a Lys at position 40 having alipophilic substituent; or (c) GLP-1 (7-36) (SEQ ID NO: 114) or GLP-1(7-37) (SEQ ID NO: 124) having at least one modification selected fromthe group consisting of: (i) substitution of D-alanine, glycine oralpha-amino isobutyric acid (Aib) for alanine at position 8 and (ii) alipophilic substituent; and Z, a peptide sequence of 4-20 amino acidunits covalently bound to X, wherein each amino acid unit in saidpeptide sequence Z is selected from the group consisting of Ala, Leu,Ser, Thr, Tyr, Asn, Gln, Asp, Glu, Lys, Arg, His, Met, Orn, and aminoacid units of the general formula I

—NH—C(R¹)(R²)—C(═O)—  (I)

wherein R¹ and R² are selected from the group consisting of hydrogen,C₁₋₆-alkyl, phenyl, and phenyl-methyl, wherein C₁₋₆-alkyl is optionallysubstituted with from one to three substituents selected from halogen,hydroxy, amino, cyano, nitro, sulfono, and carboxy, and phenyl andphenyl-methyl is optionally substituted with from one to threesubstituents selected from C₁₋₆-alkyl, C₂₋₆-alkenyl, halogen, hydroxy,amino, cyano, nitro, sulfono, and carboxy, or R¹ and R² together withthe carbon atom to which they are bound form a cyclopentyl, cyclohexyl,or cycloheptyl ring, e.g. 2,4-diaminobutanoic acid and2,3-diaminopropanoic acid. Preferably, X binds to a GLP-1 receptor anddoes not include exendin-4 or exendin-3.

Z is typically a peptide sequence of 4-20 amino acid residues, e.g., inthe range of 4-15, more preferably in the range of 4-10 in particular inthe range of 4-7 amino acid residues, e.g., of 4, 5, 6, 7, 8 or 10 aminoacid residues, where 6 amino acid residues are preferred. Preferably, Zcontains at least one Lys residue. In a preferred embodiment of theinvention each of the amino acid residues in the peptide sequence Z areindependently selected from the group consisting of Ala, Leu, Ser, Thr,Tyr, Asn, Gln, Asp, Glu, Lys, Arg, His, Met, Orn, diaminobutanoic acidand diaminopropanoic acid. Preferably, the amino acid residues areselected from Glu, Lys, and Met, especially Lys, or the amino acidresidues are selected from the group consisting of Asn, Glu and Lys. Theabove-mentioned amino acids may have either D- or L-configuration, butpreferably the above-mentioned amino acids have an L-configuration. In apreferred embodiment of the invention Z contains at least 1 lysineresidue or when Z is attached via a peptide bond to the N-terminal ofsaid peptide X then Z has an amino acid sequence selected from the groupconsisting of Asn-(Glu)n wherein n is an integer from 3 to 7.

Thus, illustrative examples of the peptide sequence Z are:

Lys-Lys-Lys-Lys(SEQ ID N0:1), Xaa-Lys-Lys-Lys, Lys-Xaa-Lys-Lys,Lys-Lys-Xaa-Lys, Lys-Lys-Lys-Xaa, Xaa-Xaa-Lys-Lys, Xaa-Lys-Xaa-Lys,Xaa-Lys-Lys-Xaa, Lys-Xaa-Xaa- Lys, Lys-Xaa-Lys-Xaa, Lys-Lys-Xaa-Xaa,Xaa-Xaa-Xaa-Lys, Xaa-Xaa-Lys-Xaa, Xaa-Lys- Xaa-Xaa, Lys-Xaa-Xaa-Xaa,Xaa-Xaa-Xaa-Xaa (SEQ ID N0:2), Lys-Lys-Lys-Lys-Lys (SEQ ID N0:3),Xaa-Lys-Lys-Lys-Lys (SEQ ID N0:4), Lys-Xaa-Lys-Lys-Lys (SEQ ID N0:5),Lys-Lys-Xaa-Lys-Lys (SEQ ID N0:6), Lys-Lys-Lys-Xaa-Lys (SEQ ID N0:7),Lys- Lys-Lys-Lys-Xaa, Xaa-Xaa-Lys-Lys-Lys, Xaa-Lys-Xaa-Lys-Lys,Xaa-Lys-Lys-Xaa-Lys, Xaa-Lys-Lys-Lys-Xaa, Lys-Xaa-Xaa-Lys-Lys,Lys-Xaa-Lys-Xaa-Lys, Lys-Xaa-Lys-Lys- Xaa, Lys-Lys-Xaa-Xaa-Lys,Lys-Lys-Xaa-Lys-Xaa, Lys-Lys-Lys-Xaa-Xaa, Lys-Lys-Xaa- Xaa-Xaa,Lys-Xaa-Lys-Xaa-Xaa, Lys-Xaa-Xaa-Lys-Xaa, Lys-Xaa-Xaa-Xaa-Lys, Xaa-Lys-Lys-Xaa-Xaa, Xaa-Lys-Xaa-Xaa-Lys, Xaa-Xaa-Lys-Lys-Xaa,Xaa-Xaa-Lys-Xaa-Lys, Xaa- Xaa-Xaa-Lys-Lys, Lys-Xaa-Xaa-Xaa-Xaa,Xaa-Lys-Xaa-Xaa-Xaa, Xaa-Xaa-Lys-Xaa-Xaa, Xaa-Xaa-Xaa-Lys-Xaa,Xaa-Xaa-Xaa-Xaa-Lys, Xaa-Xaa-Xaa-Xaa-Xaa (SEQ ID NO:8),

Lys-Lys-Lys-Lys-Lys-Lys (SEQ ID N0:9), Xaa-Lys-Lys-Lys-Lys-Lys (SEQ IDNO:10), Lys-Xaa-Lys-Lys-Lys-Lys(SEQ ID NO:11), Lys-Lys-Xaa-Lys-Lys-Lys(SEQ ID NO:12), Lys-Lys-Lys-Xaa-Lys-Lys (SEQ ID NO:13),Lys-Lys-Lys-Lys-Xaa-Lys (SEQ ID NO:14), Lys-Lys-Lys-Lys-Lys-Xaa (SEQ IDNO:15), Xaa-Xaa-Lys-Lys-Lys-Lys (SEQ ID NO:16), Xaa-Lys-Xaa-Lys-Lys-Lys(SEQ ID NO:17), Xaa-Lys-Lys-Xaa-Lys-Lys (SEQ ID NO:18),Xaa-Lys-Lys-Lys-Xaa-Lys (SEQ ID NO:19), Xaa-Lys-Lys-Lys-Lys-Xaa (SEQ IDNO:20), Lys-Xaa-Xaa-Lys-Lys-Lys (SEQ ID NO:21), Lys-Xaa-Lys-Xaa-Lys-Lys(SEQ ID NO:22), Lys-Xaa-Lys-Lys-Xaa-Lys (SEQ ID NO:23),Lys-Xaa-Lys-Lys-Lys-Xaa (SEQ ID NO:24), Lys-Lys-Xaa-Xaa-Lys-Lys (SEQ IDNO:25), Lys-Lys-Xaa-Lys-Xaa-Lys (SEQ ID N0:26), Lys-Lys-Xaa-Lys-Lys-Xaa(SEQ ID NO:27), Lys-Lys-Lys-Xaa-Xaa-Lys (SEQ ID NO:28),Lys-Lys-Lys-Xaa-Lys-Xaa (SEQ ID NO:29), Lys-Lys-Lys-Lys-Xaa-Xaa,Xaa-Xaa-Xaa-Lys- Lys-Lys, Xaa-Xaa-Lys-Xaa-Lys-Lys,Xaa-Xaa-Lys-Lys-Xaa-Lys, Xaa-Xaa-Lys-Lys-Lys- Xaa,Xaa-Lys-Xaa-Xaa-Lys-Lys, Xaa-Lys-Xaa-Lys-Xaa-Lys,Xaa-Lys-Xaa-Lys-Lys-Xaa, Xaa-Lys-Lys-Xaa-Xaa-Lys,Xaa-Lys-Lys-Xaa-Lys-Xaa, Xaa-Lys-Lys-Lys-Xaa-Xaa, Lys-Lys-Lys-Xaa-Xaa-Xaa, Lys-Lys-Xaa-Lys-Xaa-Xaa, Lys-Lys-Xaa-Xaa-Lys-Xaa,Lys-Lys- Xaa-Xaa-Xaa-Lys, Lys-Xaa-Lys-Lys-Xaa-Xaa,Lys-Xaa-Lys-Xaa-Lys-Xaa, Lys-Xaa-Lys- Xaa-Xaa-Lys,Lys-Xaa-Xaa-Lys-Lys-Xaa, Lys-Xaa-Xaa-Lys-Xaa-Lys, Lys-Xaa-Xaa-Xaa-Lys-Lys, Lys-Lys-Xaa-Xaa-Xaa-Xaa. Lys-Xaa-Lys-Xaa-Xaa-Xaa,Lys-Xaa-Xaa-Lys-Xaa- Xaa-Lys, Lys-Xaa-Xaa-Xaa-Lys-Xaa-Lys,Lys-Xaa-Xaa-Xaa-Xaa-Lys-Lys, Xaa-Lys-Lys- Xaa-Xaa-Xaa,Xaa-Lys-Xaa-Lys-Xaa-Xaa, Xaa-Lys-Xaa-Xaa-Lys-Xaa, Xaa-Lys-Xaa-Xaa-Xaa-Lys, Xaa-Xaa-Lys-Lys-Xaa-Xaa, Xaa-Xaa-Lys-Xaa-Lys-Xaa,Xaa-Xaa-Lys-Xaa-Xaa- Lys, Xaa-Xaa-Xaa-Lys-Lys-Xaa,Xaa-Xaa-Xaa-Lys-Xaa-Lys, Xaa-Xaa-Xaa-Xaa-Lys-Lys,Lys-Xaa-Xaa-Xaa-Xaa-Xaa, Xaa-Lys-Xaa-Xaa-Xaa-Xaa,Xaa-Xaa-Lys-Xaa-Xaa-Xaa, Xaa- Xaa-Xaa-Lys-Xaa-Xaa,Xaa-Xaa-Xaa-Xaa-Lys-Xaa, Xaa-Xaa-Xaa-Xaa-Xaa-Lys, Xaa-Xaa-Xaa-Xaa-Xaa-Xaa, Lys-Lys-Lys-Lys-Lys-Lys-Lys (SEQ ID NQ:30),Xaa-Lys-Lys-Lys-Lys- Lys-Lys (SEQ ID NO:31), Lys-Xaa-Lys-Lys-Lys-Lys-Lys(SEQ ID NO:32), Lys-Lys-Xaa- Lys-Lys-Lys-Lys (SEQ ID NO:33),Lys-Lys-Lys-Xaa-Lys-Lys-Lys (SEQ ID NO:34), Lys- Lys-Lys-Lys-Xaa-Lys-Lys(SEQ ID NO:35), Lys-Lys-Lys-Lys-Lys-Xaa-Lys (SEQ ID NO:36),Lys-Lys-Lys-Lys-Lys-Lys-Xaa (SEQ ID NO:37), Xaa-Xaa-Lys-Lys-Lys-Lys-Lys(SEQ ID NO:38). Xaa-Lys-Xaa-Lys-Lys-Lys-Lys (SEQ ID NO:39),Xaa-Lys-Lys-Xaa-Lys- Lys-Lys (SEQ ID NO:40), Xaa-Lys-Lys-Lys-Xaa-Lys-Lys(SEQ ID NO:41), Xaa-Lys-Lys- Lys-Lys-Xaa-Lys (SEQ ID NO:42),Lys-Xaa-Xaa-Lys-Lys-Lys-Lys (SEQ ID NO:43), Lys- Xaa-Lys-Xaa-Lys-Lys-Lys(SEQ ID NO:44), Lys-Xaa-Lys-Lys-Xaa-Lys-Lys (SEQ ID NO:45),Lys-Xaa-Lys-Lys-Lys-Xaa-Lys (SEQ ID NO:46), Lys-Lys-Xaa-Xaa-Lys-Lys-Lys(SEQ ID NO:47), Lys-Lys-Xaa-Lys-Xaa-Lys-Lys (SEQ ID NO:48),Lys-Lys-Xaa-Lys-Lys-

Xaa-lys (SEQ ID NO:49), Lys-Lys-Lys-Xaa-Xaa-Lys-Lys (SEQ ID NO:50),Lys-Lys-Lys- Xaa-Lys-Xaa-Lys (SEQ ID N0:51), Lys-Lys-Lys-Lys-Xaa-Xaa-Lys(SEQ ID NO:52), Xaa- Xaa-Xaa-Lys-Lys-Lys-Lys (SEQ ID NO:53),Xaa-Xaa-Lys-Xaa-Lys-Lys-Lys (SEQ ID NQ:54), Xaa-Xaa-Lys-Lys-Xaa-Lys-Lys(SEQ ID NO:55), Xaa-Xaa-Lys-Lys-Lys-Xaa-Lys (SEQ ID NO:56),Xaa-Lys-Xaa-Xaa-Lys-Lys-Lys (SEQ ID NO:57), Xaa-Lys-Xaa-Lys-Xaa- Lys-Lys(SEQ ID NO:58), Xaa-Lys-Xaa-Lys-Lys-Xaa-Lys (SEQ ID NO:59), Xaa-Lys-Lys-Xaa-Xaa-Lys-Lys (SEQ ID NO:60), Xaa-Lys-Lys-Xaa-Lys-Xaa-Lys (SEQ IDNO:61), Xaa- Lys-Lys-Lys-Xaa-Lys-Xaa (SEQ ID NO:62),Xaa-Lys-Lys-Xaa-Lys-Lys-Xaa (SEQ ID NO:63), Xaa-Lys-Xaa-Lys-Lys-Lys-Xaa(SEQ ID NO:64), Xaa-Lys-Lys-Lys-Xaa-Xaa-Lys (SEQ ID NO:65),Lys-Xaa-Lys-Lys-Lys-Xaa-Xaa (SEQ ID NO:66), Xaa-Lys-Lys-Lys-Lys- Xaa-Xaa(SEQ ID NO:67), Xaa-Lys-Lys-Lys-Xaa-Lys-Xaa (SEQ ID NO:68), Xaa-Lys-Lys-Lys-Xaa-Xaa-Lys (SEQ ID NO:69), Lys-Lys-Lys-Lys-Xaa-Xaa-Xaa (SEQ IDNO:70), Lys- Lys-Lys-Xaa-Xaa-Xaa-Lys (SEQ ID N0:71),Lys-Lys-Lys-Xaa-Lys-Xaa-Xaa (SEQ ID NO:72), Lys-Lys-Xaa-Lys-Lys-Xaa-Xaa(SEQ ID NO:73), Lys-Lys-Xaa-Xaa-Lys-Xaa-Lys (SEQ ID NO:74),Lys-Lys-Xaa-Xaa-Xaa-Lys-Lys (SEQ ID NO:75), Lys-Lys-Xaa-Lys-Lys- Xaa-Xaa(SEQ ID NO:76), Lys-Xaa-Lys-Lys-Xaa-Xaa-Lys (SEQ ID NO:77), Lys-Xaa-Lys-Xaa-Lys-Xaa-Lys (SEQ ID NO:78), Lys-Xaa-Lys-Xaa-Xaa-Lys-Lys (SEQ IDNO:79), Lys- Xaa-Xaa-Lys-Lys-Xaa-Lys (SEQ ID NO:80),Lys-Xaa-Xaa-Lys-Xaa-Lys-Lys (SEQ ID N0:81), Lys-Xaa-Xaa-Xaa-Lys-Lys-Lys(SEQ ID NO:82), Lys-Lys-Xaa-Xaa-Xaa-Xaa-Lys,Lys-Xaa-Lys-Xaa-Xaa-Xaa-Lys, Lys-Xaa-Xaa-Lys-Xaa-Xaa-Lys,Lys-Xaa-Xaa-Xaa-Lys- Xaa-Lys, Lys-Xaa-Xaa-Xaa-Xaa-Lys-Lys,Xaa-Lys-Lys-Xaa-Xaa-Xaa-Lys, Xaa-Lys-Xaa- Lys-Xaa-Xaa-Lys,Xaa-Lys-Xaa-Xaa-Lys-Xaa-Lys, Xaa-Lys-Xaa-Xaa-Xaa-Lys-Lys, Xaa-Xaa-Lys-Lys-Xaa-Xaa-Lys, Xaa-Xaa-Lys-Xaa-Lys-Xaa-Lys,Xaa-Xaa-Lys-Xaa-Xaa-Lys- Lys, Xaa-Xaa-Xaa-Lys-Lys-Xaa-Lys,Xaa-Xaa-Xaa-Lys-Xaa-Lys-Lys, Xaa-Xaa-Xaa-Xaa- Lys-Lys-Lys,Lys-Xaa-Xaa-Xaa-Xaa-Xaa-Lys, Xaa-Lys-Xaa-Xaa-Xaa-Xaa-Lys, Xaa-Xaa-Lys-Xaa-Xaa-Xaa-Lys, Xaa-Xaa-Xaa-Lys-Xaa-Xaa-Lys,Xaa-Xaa-Xaa-Xaa-Lys-Xaa-Lys, Xaa-Xaa-Xaa-Xaa-Xaa-Lys-Lys,Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Lys, Lys-Xaa-Xaa-Xaa-Xaa- Xaa-Xaa,Xaa-Xaa-Xaa-Xaa-Xaa-Lys-Xaa, Xaa-Lys-Xaa-Xaa-Xaa-Xaa-Xaa, Xaa-Xaa-Lys-Xaa-Xaa-Xaa, Xaa-Xaa-Xaa-Xaa-Lys-Xaa, Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa,wherein each Xaa is Independently selected from the group consisting ofAla, Leu, Ser, Thr, Tyr, Asn, Gin, Asp, Glu, Arg, His, Met, Orn, andamino acids of the formula I as defined herein, e.g.. Dbu or Dpr.

As indicated above, the amino acid residues of Z may of course all bedifferent or all be identical. However, in interesting embodiments ofthe present invention, the amino acid residues in Z are selected fromtwo or three different amino acids, or are identical amino acids.Examples of suitable peptide sequences, wherein the amino acid residuesin Z are identical are e.g., (Lys)_(n), wherein n is an integer in therange from 4 to 15, preferably in the range from 4 to 10, such as in therange from 4 to 8, e.g., in the range from about 4 to 7, e.g., Lys₄ (SEQID NO:1), Lys₅ (SEQ ID NO:2); Lys₆ (SEQ ID NO:8), Lys₇ (SEQ ID NO:30).Preferred is (Lys)₆ bound via a peptide bond to the C-terminal of X.

Examples of suitable peptide sequences, wherein the amino acid residuesin Z are selected from about two different amino acids are e.g.,(Lys-Xaa)_(m) or (Xaa-Lys)_(m), wherein m is an integer in the rangefrom about 2 to 7, preferably in the range from 2 to 5, such as in therange from 2 to 4, e.g., 3, and Xaa is independently selected from thegroup consisting of Ser, Thr, Tyr, Asn, Gln, Asp, Glu, Arg, His, Orn,2,4-diaminobutanoic acid, 2,3-diaminopropanoic acid and Met. Morepreferably such peptide sequences are e.g., (Lys-Xaa)₃ or (Xaa-Lys)₃,wherein Xaa is as defined above, such as (Lys-Glu)₃ (SEQ ID NO:83) or(Glu-Lys)₃ (SEQ ID NO:84). Other examples of suitable peptide sequences,wherein the amino acid residues in Z are selected from about two aminoacid residues are e.g., Lys_(p)-Xaa_(q) or Xaa_(p)-Lys_(q), wherein pand q are integers in the range from 1 to 14, with the proviso that p+qis in the range from 4 to 15, preferably in the range from 4 to 10, suchas in the range from 4 to 8, e.g., in the range from 4 to 6, e.g., 4, 5or 6, and Xaa is independently selected from the group consisting ofSer, Thr, Tyr, Asn, Gln, Asp, Glu, Arg, His and Met. More preferablysuch peptide sequences are e.g., Lys₃-Xaa₃ or Xaa₃-Lys₃, wherein Xaa isas defined above, such as Lys₃-Glu₃ (SEQ ID NO:85) or Glu₃-Lys₃ (SEQ IDNO:86). More preferred Z sequences consists of a sequence of amino acidresidues selected from Asn and Gln together with 4-7 amino acid residuesselected from Glu and Asp, such as Asn-(Glu)₅ (SEQ ID NO: 141),Asn-(Glu)₆ (SEQ ID NO: 142), Gln-(Glu)₅ (SEQ ID NO: 143), Asn-(Asp)₅(SEQ ID NO: 144), and Gln-(Asp)₅ (SEQ ID NO: 145), which is theN-terminal part of the peptide conjugate of the invention.

Examples of suitable peptide sequences, wherein the amino acid residuesin Z are selected from three different amino acids are e.g.,Xaa¹-(Lys)_(x)-(Xaa²)_(y), Xaa¹-(Xaa²)_(x)-(Lys)_(y),(Lys)_(x)-(Xaa²)_(y)-Xaa¹, (Xaa¹)_(x)-(Lys)_(y)-Xaa²,(Lys)_(x)-Xaa¹-(Xaa²)_(y), (Xaa¹)_(x)-Xaa²-(Lys)_(y), Xaa¹-Lys-Xaa²-Lys,Xaa¹-Lys-Xaa²-Lys-Xaa², Xaa¹-Lys-Xaa²-Lys-Xaa²-Lys, Xaa¹-Xaa²-Lys-Xaa²,Xaa¹-Xaa²-Lys-Xaa²-Lys, Xaa¹-Xaa¹-Lys-Xaa²-Lys-Xaa², Lys-Xaa²-Lys-Xaa¹,Lys-Xaa²-Lys-Xaa²-Xaa¹, Lys-Xaa²-Lys-Xaa²-Lys-Xaa¹, Xaa²-Lys-Xaa²-Xaa¹,Xaa²-Lys-Xaa²-Lys-Xaa¹, Xaa²-Lys-Xaa¹-Lys-Xaa²-Xaa¹, etc., wherein x andy are integers in the range from about 1 to 5 with the proviso that x+yis at the most 6, and Xaa¹ and Xaa² is independently selected from aboutthe group consisting of Ala, Leu, Ser, Thr, Tyr, Asn, Gln, Asp, Glu,Arg, His, Met, Orn, 2,3-diaminopropanoic acid, 2,4-diaminobutanoic acidand amino acids of the formula I as defined herein.

In preferred embodiments of the invention the ratio between the minimumeffective oral dose of said peptide conjugate and the minimum effectivedose of the peptide, X is at least 1:5.

A most preferred embodiment of the invention is directed to a novelpeptide conjugate comprising a peptide X being an agonist of GLP-1and/or exendin-4 activity selected from the group consisting of

des Pro³⁶-exendin-4(1-39)-NH₂ (SEQ ID NO:101),

des Pro³⁶-exendin-4(1-40)-NH₂ (SEQ ID NO: 139),

des Pro³⁶-des Pro³⁷-exendin-4(1-39)-NH₂ (SEQ ID NO: 130),

des Pro³⁶-des Pro³⁷-des Pro³⁸-exendin-4(1-39)-NH₂ (SEQ ID NO: 132),

des Pro³⁶-des Pro³⁷-des Pro³⁸-exendin-4(1-40)-NH₂ (SEQ ID NO: 140),

des Ala³⁵-exendin-4(1-39)-NH₂ (SEQ ID NO:105),

des Gly³⁴-exendin-4(1-39)-NH₂ (SEQ ID NO:106),

des Gly³⁴-(Lys⁴⁰(palmitoyl))exendin-4(1-39)-NH₂ (SEQ ID NO:108),

des Ala³⁵-(Lys⁴⁰(palmitoyl))exendin-4(1-39)-NH₂ (SEQ ID NO:109),

des Pro³⁶-(Lys⁴⁰(palmitoyl))exendin-4(1-39)-NH₂ (SEQ ID NO:110),

Compound (iii) Gly⁸-GLP-1(7-36)-NH₂ (SEQ ID NO: 87), Gly⁸-GLP-1(7-37)(SEQ ID NO: 123), and Gly⁸-GLP-1(7-36)-Lys³⁷(palmitoyl)-NH₂ (SEQ ID NO:147), and being C-terminally bound via a peptide bond to a peptidesequence Z selected from the group consisting of (Lys)n where n is aninteger from 4 to 8, preferably n is 6.

It should be understood that the peptide conjugates of the inventionmight also be in the preferred amide (NH₂) or in the free acid (OH) formor in the form of a salt thereof. Exemplary peptide conjugates of theinvention are

Gly⁸-GLP-1 (7-36)-Lys₆-NH₂ (SEQ ID NO:88),

(Gly⁸,Lys³⁷(palmitoyl)-GLP-1(7-36)(Human)-Lys₇-NH₂ (SEQ ID NO:89),

des Ser³⁹-exendin-4(1-39)-(Lys)₆-NH₂ (SEQ ID NO:91),

exendin-4(1-39)-Lys₆-NH₂ (SEQ ID NO:92),

des Pro³⁶-exendin-4(1-39)-Lys₆-NH₂ (SEQ ID NO:93),

des Ala³⁵-exendin-4(1-39)-Lys₆-NH₂ (SEQ ID NO:94),

des Gly³⁴-exendin-4(1-39)-Lys₆-NH₂ (SEQ ID NO:95),

des Ser³⁹-(Lys⁴⁰(palmitoyl))exendin-4(1-39)-Lys₇-NH₂ (SEQ ID NO:96),

des Gly³⁴-(Lys⁴⁰(palmitoyl))exendin-4(1-39)-Lys₇-NH₂ (SEQ ID NO:97),

des Ala³⁵-(Lys⁴⁰(palmitoyl))exendin-4(1-39)-Lys₇-NH₂ (SEQ ID NO:98),

des Pro³⁶-(Lys⁴⁰(palmitoyl))exendin-4(1-39)-Lys₇-NH₂ (SEQ ID NO:99),

Lys⁴⁰(palmitoyl)exendin-4(1-39)-Lys₇-NH₂ (SEQ ID NO100),

des Pro³⁶, Pro³⁷-exendin-4(1-39)-Lys₆-NH₂ (SEQ ID NO: 131),

Lys₆-des Pro³⁶, Pro³⁷, Pro³⁸-exendin-4(1-39)-NH₂ (SEQ ID NO: 134)

Asn(Glu)₅-des Pro³⁶, Pro³⁷, Pro³⁸-exendin-4(1-39)-NH₂ (SEQ ID NO: 137),

Lys₆-des Pro³⁶, Pro³⁷, Pro³⁸-exendin-4(1-39)-Lys₆-NH₂ (SEQ ID NO: 135),

Asn(Glu)₅-des Pro³⁶, Pro³⁷, Pro³⁸-exendin-4(1-39)-Lys₆-NH₂(SEQ ID NO:136),

des Pro³⁶, Pro³⁷, Pro³⁸-exendin-4(1-39)-Lys₆-NH₂ (SEQ ID NO: 133),

Ser⁸-GLP-1 (7-36)-Lys₆-NH₂ (SEQ ID NO: 115),

Aib⁸-GLP-1 (7-36)-Lys₆-NH₂ (SEQ ID NO: 116),

Lys₆-Gly⁸-GLP-1 (7-36)-Lys₆-NH₂ (SEQ ID NO: 118),

Lys₆-Gly⁸-GLP-1 (7-36)-NH₂ (SEQ ID NO: 119),

(Gly⁸,Lys²⁶(palmitoyl)-GLP-1(7-36)(Human)-Lys₆-NH₂ (SEQ ID NO: 103),

(Gly⁸,Lys³⁴(palmitoyl)-GLP-1(7-36)(Human)-Lys₆-NH₂ (SEQ ID NO: 90),

Gly⁸-GLP-1 (7-36)-Lys₈-NH₂ (SEQ ID NO: 120),

Gly⁸-GLP-1 (7-36)-Lys₁₀-NH₂ (SEQ ID NO: 121),

Gly⁸-GLP-1 (7-37)-Lys₆-NH₂ (SEQ ID NO: 122),

and the free acid thereof and a pharmaceutically acceptable saltthereof.

Among the Preferred conjugates are

des Pro³⁶-exendin-4(1-39)-Lys₆-NH₂ (SEQ ID NO:93),

Gly⁸-GLP-1 (7-36)-Lys₆-NH₂ (SEQ ID NO:88),

des Pro³⁶, Pro³⁷, Pro³⁸-exendin-4(1-39)-Lys₆-NH₂ (SEQ ID NO: 133), and

their salts as defined herein.

In a most specific embodiment, the conjugates are selected from thegroup consisting of Gly⁸-GLP-1-(7-36)(Human)-NH₂ (SEQ ID NO: 87),Gly⁸-GLP-1-(7-36)(Human)-Lys₆-NH₂ (SEQ ID NO: 88),Gly⁸Lys³⁷(palmitoyl)-GLP-1-(7-36)(Human)-Lys₇-NH₂ (SEQ ID NO: 89),Gly⁸Lys³⁴(palmitoyl)-GLP-1-(7-36)(Human)3-Lys₆-NH₂ (SEQ ID NO: 90), desSer³⁹-exendin-4(1-39)-Lys₆-NH₂ (SEQ ID NO: 91), exendin-4(1-39)-Lys₆-NH₂(SEQ ID NO: 92), des Pro³⁶-exendin-4(1-39)-Lys₆-NH₂ (SEQ ID NO: 93), desAla³⁵-exendin-4(1-39)-Lys₆-NH₂ (SEQ ID NO: 94), desGly³⁴-exendin-4(1-39)-Lys₆-NH₂ (SEQ ID NO: 95), desSer³⁹-(Lys⁴⁰(palmitoyl))exendin-4(1-39)-Lys₇-NH₂ (SEQ ID NO: 96), desGly³⁴-(Lys⁴⁰(palmitoyl))exendin-4(1-39)-Lys₇-NH₂ (SEQ ID NO: 97), desAla³⁵-(Lys⁴⁰(palmitoyl))exendin-4(1-39)-Lys₇-NH₂ (SEQ ID NO: 98), desPro³⁶-(Lys⁴⁰(palmitoyl))exendin-4(1-39)-Lys₇-NH₂ (SEQ ID NO: 99) andLys⁴⁰(palmitoyl)exendin-4(1-39)-Lys₇-NH₂ (SEQ ID NO: 100).

The provision of the peptide conjugates of the present invention enablesblood glucose lowering peptides, such as GLP-1 and exendins and theiractive analogues to be administered orally. The herein preferredterminal peptide fragments Z are chosen so as to induce an alpha-helicalstructure to the peptide X without significantly affecting the desiredactivity of X. Said helical structure stabilises the peptide chain, e.g.againsts degradation, as evidenced by the increased half life of from 2to 3 times of the conjugated peptide compared to the unconjugatedpeptide, cf. table 5 below. The peptide sequence Z is the part of thepeptide conjugate responsible for introducing of a certain structureinto the molecule so that the minimum effective dose is lowered at leastfive fold. Preferably the minimum effective dose is lowered at least tenfold, more preferably 25 fold, even more preferably 40 fold, and mostpreferably 50 fold. Therefore, the present invention also relates to theuse of a peptide sequence (Z) as defined above for the preparation of asaid peptide conjugate as defined above.

Thus, the invention also relates to a novel peptide conjugate comprisinga peptide X as defined herein and wherein X reduces the blood glucoselevel in a mammal where the ratio between the minimum effective oraldose of said peptide conjugate and the minimum effective oral dose ofthe peptide X is at least 1:5.

Specifically, the invention is directed to a method for stimulatinginsulin release in a mammal comprising administering an effectiveinsulinotropic amount of the peptide conjugate of the present invention,a method of lowering blood glucose level in a mammal comprisingadministering an amount of the peptide conjugate of the presentinvention effective to lower blood glucose level in said mammal, amethod of reducing gastric motility in a mammal in an amount of thepeptide conjugate of the present invention effective to reduce gastricmotility, a method of delaying gastric emptying in a mammal in an amountof the peptide conjugate of the present invention effective to delaygastric emptying, a method of inhibiting food uptake in a mammal in anamount of the peptide conjugate of the present invention effective toinhibit food uptake and a method of lowering plasma lipid level in amammal comprising administering an amount of peptide conjugate of thepresent invention effective to lower plasma lipid level in said mammal.Specifically, the peptide conjugate of the present invention may be usedin treatment of diabetes type 1 or type 2, obesity, eating disorders,hyperglycemia, metabolic disorders, gastric disease and insulinresistance syndrome.

The present invention also relates to methods for the preparation ofsaid peptide conjugate, by means of recombinant DNA technologycomprising the steps of (a) introducing a nucleic acid sequence encodingsaid conjugate into a host cell and (b) culturing said host cell and (c)isolating said conjugate from the culture or (a) culturing a recombinanthost cell comprising a nucleic acid sequence encoding said conjugateunder conditions permitting the production of said conjugate and (b)isolating said conjugate from the culture.

The method also relates to methods for the preparation of said peptideconjugate in which peptide X is obtained via recombinant DNA methods byisolating said peptide. X is then conjugated to Z which is attached to asolid support or has been prepared by solid phase synthetic methods.Furthermore, the invention relates to the preparation of the peptideconjugate of the present invention by peptide synthetic methods.Furthermore, the invention relates to the preparation of the peptideconjugate of the present invention by peptide synthetic methods.

The conjugates of the invention comprising an N-terminal sequence offrom 33 to 39, preferably from 36 to 38, amino acid residues having asubstantial homology to the native exendin-4 N-terminal sequence thoughtto be essential for receptor binding (insulinotropic activity) and aC-terminal sequence Z possess as a further advantage improved stabilitycompared to native exendins and C-terminally truncated forms of exendin.Likewise, the GLP-1 peptide conjugate Compound 4 shows improvedstability compared to the unconjugated Compound (iii).

Compositions

The invention also concerns a composition comprising the exendin variantor the peptide conjugate of the present invention in combination with aphysiologically acceptable carrier. Such compositions may be in a formadapted to oral, parenteral (including subcutaneous (s.c.), intravenous(i.v.), intramuscular (i.m.), epidural, direct brain and intraperitoneal(i.p.)), rectal, intratracheal, intranasal, dermal, vaginal, buccal,ocularly, or pulmonary administration, preferably in a form adapted tosubcutaneous or oral administration, and such compositions may beprepared in a manner well-known to the person skilled in the art, e.g.,as generally described in “Remington's Pharmaceutical Sciences”, 17. Ed.Alfonso R. Gennaro (Ed.), Mark Publishing Company, Easton, Pa., U.S.A.,1985 and more recent editions and in the monographs in the “Drugs andthe Pharmaceutical Sciences” series, Marcel Dekker. The compositions mayappear in conventional forms, for example, capsules, tablets, aerosols,topical application forms, liquid or semiliquid forms, such assolutions, suspensions, dispersions, emulsions, micelles or liposomes.Preferred are liquid compositions suitable for s.c. administration. In apreferred embodiment, the compositions of the present invention areadministered subcutaneously. In an alternative preferred embodiment, thecompositions of the present invention are administered orally, and insuch cases one preferred administration form is a tablet or capsule.

The pharmaceutical carrier or diluent employed may be a conventionalsolid or liquid carrier. Examples of solid carriers are lactose, terraalba, sucrose, cyclodextrin, talc, gelatin, agar, pectin, acacia,magnesium stearate, stearic acid or lower alkyl ethers of cellulose.Examples of liquid carriers are syrup, peanut oil, olive oil,phospholipids, sterols, fatty acids, fatty acid amines, polyoxyethylene,isotonic buffer solutions and water. Similarly, the carrier or diluentmay include any sustained release material known in the art, such asglyceryl monostearate or glyceryl distearate, alone or mixed with a wax.If a solid carrier is used for oral administration, the preparation maybe tabletted, placed in a hard gelatin capsule in powder or pellet formor it can be in the form of a troche or lozenge. The amount of solidcarrier will vary widely but will usually be from about about 25 mg toabout 1 g.

A typical tablet which may be prepared by conventional tablettingtechniques may contain:

-   -   Core: active compound (as free compound of the invention or salt        thereof) 100 mg; colloidal silicon dioxide (Aerosil) 1.5 mg;        cellulose, microcryst. (Avicel) 70 mg; modified cellulose gum        (Ac-Di-Sol) 7.5 mg; magnesium stearate.    -   Coating: HPMC approx. 9 mg; *Mywacett 9-40 T approx. 0.9 mg;        *acylated monoglyceride used as plasticizer for film coating.

If a liquid carrier is used, the preparation may be in the form of asyrup, emulsion, soft gelatin capsule or sterile injectable liquid suchas an aqueous or non-aqueous liquid suspension or solution.

For nasal administration, the preparation may contain a compound of thepresent invention, preferably a conjugate, dissolved or suspended in aliquid carrier, in particular, an aqueous carrier, for aerosolapplication. The carrier may contain additives such as solubilizingagents, e.g., propylene glycol, surfactants such as bile acid salts orpolyoxyethylene higher alcohol ethers, absorption enhancers such aslecithin (phosphatidylcholine) or cyclodextrin, or preservatives such asparabines.

The composition may also be in a form suited for local or systemicinjection or infusion and may, as such, be formulated with sterile wateror an isotonic saline or glucose solution. The compositions may besterilized by conventional sterilization techniques which are well knownin the art. The resulting aqueous solutions may be packaged for use orfiltered under aseptic conditions and lyophilized, the lyophilizedpreparation being combined with the sterile aqueous solution prior toadministration. Preferably, the formulation to be used for intravenous,subcutaneous and oral dosing will be a solution of the active compoundin buffer. The preparation may be produced immediately before use fromactive drug substance and sterile buffer solution. One preferred methodof sterilization may be by sterile filtration of a solution madeimmediately prior to use. The composition may contain pharmaceuticallyacceptable auxiliary substances as required to approximate physiologicalconditions, such as buffering agents, tonicity adjusting agents and thelike, for instance sodium acetate, sodium lactate, sodium chloride,potassium chloride, calcium chloride, etc.

The compounds of the invention possess valuable pharmacologicalproperties, e.g. stability towards proteolytic enzymes. In vitrostability studies with the present peptides and peptide conjugates inthe presence of selected proteolytic enzymes show increased half livesof the novel peptides compared to prior art peptides. Thus, thecompounds of the invention exhibit considerably extended duration ofaction in vivo compared to GLP-1 and other GLP-1 agonists. Furthermore,the compounds of the invention stimulate cAMP formation. This effect maybe demonstrated in a cAMP assay, e.g. as described in WO 98/08871.

The peptide compounds of the present invention are agonists of GLP-1activity and/or exendin-4 activity and improves blood glucose tolerancein diabetic mammals as determined by assays known in the art for aparticular peptide. Examples of such an assay are described herein.Thus, the invention also concerns the exendin variants and peptideconjugates as defined above for use in therapy, and the use of thepeptide conjugates as defined above for the manufacture of apharmaceutical composition for use in therapy, e.g., in the treatment ofdiabetes type 1 or type 2, obesity, eating disorders and insulinresistance syndrome.

In specific embodiments, the exendin variants and peptide conjugates ofthe invention may be used to stimulate insulin release, lower bloodglucose level, reduce gastric motility, delay gastric emptying, inhibitfood uptake, e.g. by suppression of appetite, or lower the plasma lipidlevel in a vertebrate or a mammal. The novel compounds of the inventionmay also be used generally in the treatment of diabetes mellitusassociated with a risk for hyperglycemia, i.e. where insulin sensitivityis decreased with stress, myocardia infection, stroke and infections, orin cases of insulin resistance during pregnancy. The novel compounds mayalso be used in the treatment of other types of diabetes, such as caseswhere diabetes may be secondary to other endocrine diseases such asacromegaly, Cushing's syndrome, pheochromocytoma, glucagonoma,somatostatinoma, primary aldosteronism, or secondary to administrationof certain hormones causing hyperglycemia, or secondary to certain drugs(antihypertensive drugs, thiazide diuretics, preparations containingestrogen, psychoactive drugs, sympathomimetic agents. Furthermore, thenovel compounds of the invention may be used generally in the treatmentof diseases and conditions associated with a risk for hypoglycemia, i.e.where endogenous glucose production is decreased, as following alcoholingestion, or in cases where the sensitivity to insulin is increased inpatients with hypopituitarism or primary adrenocortical insufficiency,or where insulin clearance is decreased as with progressive renalinsufficiency.

Other specific therapeutic uses are described in WO 99/40788 (relatingto the inotropic and diuretic effects of exendin and GLP-1) WO 98/39022(relating to a method of sedating a mammalian subject having increasedactivation of the central or peripheral nervous system comprisingadministering exendin or GLP-1 or an agonist of exendin or GLP-1 to thesubject to produce a sedative or anxiolytic effect on the subject), WO93/18786 (relating to the treatment of diabetes using GLP-1(7-37) (SEQID NO: 124) or GLP-1(7-36)amide (SEQ ID NO: 114) in a regimen whichadditionally comprises treatment with an oral hypoglycaemic agent, suchas sulfonylurea, producing a strong synergistic effect), WO 98/19698(relating to the use of GLP-1 analogs for the regulation of obesity), WO98/08531 (relating to the use of GLP-1 or analogs in a method ofreducing mortality and morbidity after myocardial infarction), WO98/08873 (relating to the use of GLP-1 or analogs in a method ofattenuating post-surgical catabolic changes and hormonal responses tostress). Besides, the compounds of the invention are suitable in acombination therapy with other antidiabetic agents, such as insulin,metformin, sulfonyl ureas and thiazolidinediones, or in combinationtherapy with other antiobesity agents, such as leptin, dexphenfluramine,amphetam in etc.

Definitions

A “peptide” as used herein is any compound produced by amide formationbetween a carboxyl group of one amino acid and an amino group ofanother. The amide bonds in peptides may be called peptide bonds. Theword peptide usually applies to compounds whose amide bonds are formedbetween C-1 of one amino acid and N-2 of another (sometimes calledeupeptide bonds), but it includes compounds with residues linked byother amide bonds (sometimes called isopeptide bonds). Peptides withfewer than about 10-20 residues may also be called oligopeptides; thosewith more, polypeptides. Polypeptides of specific sequence of more thanabout 50 residues are usually known as proteins. A “natural polypeptidesequence” as used herein refers to a polypeptide sequence consisting ofnatural L-amino acid residues and which is capable of being expressed bya recombinant host cell. The X compounds herein are all peptidesequences of 40 amino acid residues or less.

“GLP-1” as used herein includes GLP-1(7-37)-OH (SEQ ID NO: 124),GLP-1(7-37)-NH₂ (SEQ ID NO: 124), GLP-1(7-36)-OH (SEQ ID NO: 114), andGLP-1(7-36)-NH₂ (SEQ ID NO: 114).

“Agonist” refers to an endogenous substance or a drug that can interactwith a receptor and initiate a physiological or a pharmacologicalresponse characteristic of that receptor (contraction, relaxation,secretion, enzyme activation, etc.).

“Antagonist” refers to a drug or a compound that opposes thephysiological effects of another. At the receptor level, it is achemical entity that opposes the receptor-associated responses normallyinduced by another bioactive agent.

“Partial agonist” refers to an agonist which is unable to induce maximalactivation of a receptor population, regardless of the amount of drugapplied. A “partial agonist” may be termed “agonist with intermediateintrinsic efficacy” in a given tissue. Moreover, a partial agonist mayantagonize the effect of a full agonist that acts on the same receptor.

“Receptor” refers to a molecule or a polymeric structure in or on a cellthat specifically recognizes and binds a compound acting as a molecularmessenger (neurotransmitter, hormone, lymphokine, lectin, drug, etc.).

By “exendin variant” of the present invention is to be understood avariant of a parent exendin peptide having at least about 90% homologyto exendin-4 and most preferably having at least about 95% homology toexendin-4(1-39) (SEQ ID NO: 102), which has exendin activity, e.g.,lowers the blood glucose level in a mammal and binds to a GLP-1receptor. “Exendin-4” as used herein refers to exendin-4(1-39) (SEQ IDNO: 102) the amino acid sequence of which is disclosed in U.S. Pat. No.5,424,286, SEQ ID NO:2, and exendin-4(1-40) (SEQ ID NO: 138) asdisclosed by Chen & Drucker in The Journal of Biological Chemistry, Vol.272, No. 7, pp. 4108-15 which differs only in having glycine in position40 as C-terminal amino acid residue. The homology of the parent exendinis determined as the degree of identity between two protein sequencesindicating a derivation of the first sequence from the second. Thehomology may suitably be determined by means of computer programs knownin the art such as GAP provided in the GCG program package (ProgramManual for the Wisconsin Package, Version 8, August 1994, GeneticsComputer Group, 575 Science Drive, Madison, Wis., USA 53711) (Needleman,S. B. and Wunsch, C. D., (1970), J. Mol. Biol. 48:443-453). Thefollowing settings for polypeptide sequence comparison may be used: GAPcreation penalty of 3.0 and GAP extension penalty of 0.1.

“Salts” include pharmaceutically acceptable salts, such as acid additionsalts and basic salts. Examples of acid addition salts are hydrochloridesalts, sodium salts, hydrobromide salts, etc. Examples of basic saltsare salts where the cation is selected from alkali metals, such assodium and potassium, alkaline earth metals, such as calcium, andammonium ions ⁺N(R³)₃(R⁴), where R³ and R⁴ independently designatesoptionally substituted C₁₋₆-alkyl, optionally substituted C₂₋₆-alkenyl,optionally substituted aryl, or optionally substituted heteroaryl. Otherexamples of pharmaceutically acceptable salts are; e.g., those describedin “Remington's Pharmaceutical Sciences” 17. Ed. Alfonso R. Gennaro(Ed.), Mark Publishing Company, Easton, Pa., U.S.A., 1985 and morerecent editions, and in Encyclopedia of Pharmaceutical Technology.

Preparation of Variants and Conjugates

The exendin variants and the peptide conjugates of the invention may beprepared by methods known per se in the art. Thus, the variants and thepeptide sequences X and Z may be prepared by standardpeptide-preparation techniques such as solution synthesis orMerrifield-type solid phase synthesis. It is believed that the Boc(tert.butyloxycarbonyl) as well as the Fmoc(9-fluorenylmethyloxycarbonyl) strategies are applicable.

In one possible synthesis strategy, the peptide conjugates of theinvention may be prepared by solid phase synthesis by first constructingthe peptide sequence Z using well-known standard protection, couplingand deprotection procedures, thereafter sequentially coupling thepeptide sequence X on Z in a manner similar to the construction of Z,and finally cleaving off the entire peptide conjugate from the carrier.This strategy yields a peptide conjugate, wherein the peptide sequence Zis covalently bound to the peptide X at the C-terminal carbonyl functionof X. If the desired peptide conjugate, however, is a peptide conjugate,wherein two stabilising sequences Z are covalently and independentlybound to both the C- and the N-terminal of the peptide X, the abovestrategy is also applicable but, as will be understood by the personskilled in the art, before cleaving the off the C-terminal bound peptideconjugate from the solid support, it is necessary to sequentially couplethe second peptide sequence Z to the N-terminal of X in a manner similarto the procedure described above. This strategy may also be used toattach Z to the carbonyl function on the side chain of Glu or Asp. Apossible strategy for the preparation of peptide conjugates, wherein thepeptide sequence Z is covalently bound to the N-terminal nitrogen atomor covalently bound to the nitrogen atom on the side chain of Lys, Argor His of X is analogous with the method described above, i.e. saidpeptide conjugates may be prepared by solid phase synthesis by firstconstructing the peptide sequence X using well-known standardprotection, coupling and deprotection procedures, thereaftersequentially coupling the peptide sequence Z on X in a manner similar tothe construction of X, and finally cleaving off the entire peptideconjugate from the carrier. Another possible strategy is to prepare oneor both of the two sequences X and Z (or parts thereof) separately bysolution synthesis, solid phase synthesis, recombinant techniques, orenzymatic synthesis, followed by coupling of the two sequences bywell-known segment condensation procedures, either in solution or usingsolid phase techniques or a combination thereof. In one embodiment, Xmay be prepared by recombinant DNA methods and Z may be prepared bysolid phase synthesis. The conjugation of X and Z may be carried out byusing chemical ligation. This technique allows for the assembling oftotally unprotected peptide segments in a highly specific manner (Liu etal., 1996, J. Am. Chem. Soc. 118:307-312 and Dawson et al., 1996,226:776). The conjugation can also be performed by protease-catalysedpeptide bond formation, which offers a highly specific technique tocombine totally unprotected peptide segments via a peptide bond (W.Kullmann, 1987, Enzymatic Peptide Synthesis, CRC Press, Boca Raton,Fla., pp. 41-59).

Side chain derivatization of Lys, Arg, His, Trp, Ser, Thr, Cys, Tyr, Aspand Glu with the peptide sequence, Z, can be carried out by traditionalconvergent peptide synthesis using suitable orthogonal protectingschemes as known in the art, or by using the equally well known generalsolid phase method with suitable orthogonal removable chain protection.

Furthermore, it is envisaged that a combination of the above-mentionedstrategies may be especially applicable where a modified peptidesequence, e.g., from a peptide X comprising isosteric bonds such asreduced peptide bonds, is to be coupled to a peptide sequence Z. In thiscase, it may be advantageous to prepare the immobilised fragment of Z bysuccessive coupling of amino acids, and then couple a complete peptidesequence X (prepared in solution or fully or partially using solid phasetechniques or by means of recombinant techniques) to the fragment.

Examples of suitable solid support materials (SSM) are e.g.,functionalised resins such as polystyrene, polyacrylamide,polydimethylacrylamide, polyethyleneglycol, cellulose, polyethylene,polyethyleneglycol grafted on polystyrene, latex, dynabeads, etc. Itshould be understood that it may be necessary or desirable that theC-terminal amino acid of the peptide sequence Z or the C-terminal aminoacid of the peptide X is attached to the solid support material by meansof a common linker such as 2,4-dimethoxy-4′-hydroxy-benzophenone,4-(4-hydroxy-methyl-3-methoxyphenoxy)-butyric acid,4-hydroxy-methyl-benzoic acid, 4-hydroxymethyl-phenoxyacetic acid,3-(4-hydroxymethylphenoxy)propionic acid, andp-[(R,S)-a[1-(9H-fluoren-9-yl)methoxyformamido]-2,4-dimethoxybenzyl]-phenoxy-aceticacid.

The variants and the peptide conjugates of the invention may be cleavedfrom the solid support material by means of an acid such astrifluoracetic acid, trifluoromethanesulfonic acid, hydrogen bromide,hydrogen chloride, hydrogen fluoride, etc. optionally in combinationwith one or more “scavengers” suitable for the purpose, e.g.,ethanedithiol, triisopropylsilane, phenol, thioanisole, etc., or thepeptide conjugate of the invention may be cleaved from the solid supportby means of a base such as ammonia, hydrazine, an alkoxide, such assodium ethoxide, an hydroxide, such as sodium hydroxide, etc.

Thus, the present invention also relates to a method for the preparationof a pharmacologically active peptide conjugate, wherein Z is covalentlybound to X, preferably via a peptide bond. A method for the preparationof a peptide conjugate of formula I (X—Z), comprises the steps of:

a) coupling an amino acid or dipeptide having suitable protectinggroups, including an N-α-protecting group, in the activated form to animmobilised peptide sequence H—Z—SSM, thereby forming an immobilisedN-α-protected peptide fragment,

b) removing said N-α-protecting group, thereby forming an immobilisedprotected peptide fragment having an unprotected N-terminal,

c) coupling an additional amino acid or dipeptide having suitableprotecting groups including an N-α-protecting group in the carboxylactivated form to the N-terminal of the immobilised peptide fragment,and repeating the removal/coupling step procedure in step b) and c)until the desired peptide sequence X is obtained, and then

d) cleaving off the peptide conjugate from the solid support material.

A method for the preparation of a peptide conjugate of formula II (Z—X),comprises the steps of:

a) coupling an amino acid or dipeptide having suitable protectinggroups, including an N-α-protecting group, in the activated form to asolid support material (SSM), thereby forming an immobilised protectedamino acid or a protected dipeptide,

b) removing said N-α-protecting group, thereby forming an immobilisedamino acid or peptide fragment having an unprotected N-terminal,

c) coupling an additional amino acid or dipeptide having suitableprotecting groups, including an N-α-protecting group, in the carboxylactivated form to the N-terminal of the immobilised amino acid orpeptide fragment, and repeating the removal/coupling step procedure instep b) and c) until the desired peptide sequence X is obtained,

d) coupling an additional amino acid or dipeptide having suitableprotecting groups, including an N-α-protecting group, in the carboxylactivated form to the N-terminal of the immobilised peptide fragment,and repeating the removal/coupling step procedure in step b) and d)until the desired peptide sequence Z is obtained, and then

e) cleaving off the peptide conjugate from the solid support material.

Furthermore, a method for the preparation of a peptide conjugate offormula III (Z—X—Z), comprises the steps of:

a) coupling an amino acid or dipeptide having suitable protectinggroups, including an N-α-protecting group, in the carboxyl activatedform to an immobilised peptide sequence H—Z—SSM, thereby forming animmobilised N-α-protected peptide fragment,

b) removing said N-α-protecting group, thereby forming an immobilisedpeptide fragment having an unprotected N-terminal,

c) coupling an additional amino acid or dipeptide having suitableprotecting groups, including an N-α-protecting group, in the carboxylactivated form to the N-terminal of the immobilised peptide fragment,and repeating the removal/coupling step procedure in step b) and c)until the desired peptide sequence X is obtained, and then

d) coupling an additional amino acid or dipeptide having suitableprotecting groups, including an N-α-protecting group, in the carboxylactivated form to the N-terminal of the immobilised peptide fragment,and repeating the removal/coupling step procedure in step b) and d)until the desired peptide sequence Z is obtained, and then

e) cleaving off the peptide conjugate from the solid support material.

The coupling, removal and cleavage steps are performed by methods knownto the person skilled in the art taking into consideration theprotection strategy and the selected solid phase material. In general,however, it is believed that the Boc (tert.butyloxycarbonyl) as well asthe Fmoc (9-fluorenylmethyloxycarbonyl) protection strategies areapplicable and that peptide bonds may be formed using the variousactivation procedures known to the person skilled in the art, e.g., byreacting a C-terminal activated derivative (acid halide, acid anhydride,activated ester e.g., HObt-ester, etc.) of the appropriate amino acid orpeptide with the amino group of the relevant amino acid or peptide asknown to a person skilled in peptide chemistry. Furthermore, it may benecessary or desirable to include side-chain protection groups whenusing amino acid residues carrying functional groups which are reactiveunder the prevailing conditions. The necessary protection scheme will beknown to the person skilled in the art (cf., e.g., M. Bodanszky and A.Bodanszky, “The Practice of Peptide Synthesis”, 2. Ed, Springer-Verlag,1994, J. Jones, “The Chemical Synthesis of Peptides”, Clarendon Press,1991, and Dry-land et al., 1986, J. Chem. Soc., Perkin Trans.1:125-137).

The peptides and peptide conjugates of the invention may also beprepared by means of recombinant DNA technology using general methodsand principles known to the person skilled in the art. A nucleic acidsequence encoding the peptides and peptide conjugates may be preparedsynthetically by established standard methods, e.g., the phosphoamiditemethod described by S. L. Beaucage and M. H. Caruthers, TetrahedronLetters 22, 1981, pp. 1859-1869, or the method described by Matthes etal., EMBO Journal 3, 1984, pp. 801-805. According to the phosphoamiditemethod, oligonucleotides are synthesized, e.g., in an automatic DNAsynthesizer, purified, annealed, ligated and cloned in suitable vectors.The techniques used to isolate or clone a nucleic acid sequence encodingpeptide X are known in the art and include isolation from genomic DNA,preparation from cDNA, or a combination thereof. The cloning of thenucleic acid sequences of the present invention from such genomic DNAcan be effected, e.g., by using the well known polymerase chain reaction(PCR) or antibody screening of expression libraries to detect cloned DNAfragments with shared structural features. See, e.g., Innis et al.,1990, A Guide to Methods and Application, Academic Press, New York.Other nucleic acid amplification procedures such as ligase chainreaction (LCR), ligated activated transcription (LAT) and nucleic acidsequence-based amplification (NASBA) may be used. It can then be ligatedto a nucleic acid sequence encoding Z.

The nucleic acid sequence encoding the peptides and peptide conjugatesis then inserted into a recombinant expression vector which may be anyvector which may conveniently be subjected to recombinant DNAprocedures. The choice of vector will often depend on the host cell intowhich it is to be introduced. Thus, the vector may be an autonomouslyreplicating vector, i.e., a vector which exists as an extrachromosomalentity, the replication of which is independent of chromosomalreplication, e.g., a plasmid. Alternatively, the vector may be onewhich, when introduced into a host cell, is integrated into the hostcell genome and replicated together with the chromosome(s) into which ithas been integrated.

In the vector, the nucleic acid sequence encoding the peptides andpeptide conjugates of the present invention should be operably connectedto a suitable promoter sequence. The promoter may be any nucleic acidsequence which shows transcriptional activity in the host cell of choiceand may be derived from genes encoding proteins either homologous orheterologous to the host cell. Examples of suitable promoters fordirecting the transcription of the nucleic acid sequence encoding saidpeptides and peptide conjugates in mammalian cells are the SV 40promoter (Subramani et al., Mol. Cell Biol. 1, 1981, pp. 854-864), theMT-1 (metallothionein gene) promoter (Palmiter et al., Science 222,1983, pp. 809-814) or the adenovirus 2 major late promoter, a Roussarcoma virus (RSV) promoter, cytomegalovirus (CMV) promoter (Boshart etal., 1981, Cell 41:521-530) and a bovine papilloma virus promoter (BPV).A suitable promoter for use in insect cells is the polyhedrin promoter(Vasuvedan et al., FEBS Lett. 311, 1992, pp. 7-11).

Examples of suitable promoters for directing the transcription of thenucleic acid sequence encoding the peptides and peptide conjugates,especially in a bacterial host cell, are the promoters obtained from theE. coli lac operon, the Streptomyces coelicolor agarase gene (dagA), theBacillus subtilis levansucrase gene (sacB), the Bacillus licheniformisalpha-amylase gene (amyL), the Bacillus stearothermophilus maltogenicamylase gene (amyM), the Bacillus amyloliquefaciens alpha amylase gene(amyQ), the Bacillus licheniformis penicillinase gene (penP), theBacillus subtilis xylA and xylB genes, and the prokaryoticbeta-lactamase gene (Villa-Kamaroff et al., 1978, Proceedings of theNational Academy of Sciences USA 75:3727-3731), as well as the tacpromoter (DeBoer et al., 1983, Proceedings of the National Academy ofSciences USA 80:21 25). Further promoters are described in “Usefulproteins from recombinant bacteria” in Scientific American, 1980,242:74-94; and in Sambrook et al., 1989, supra. Examples of suitablepromoters for directing the transcription of the nucleic acid sequenceencoding the peptides and peptide conjugates in a filamentous fungalhost cell are promoters obtained from the genes encoding Aspergillusoryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillusniger neutral alpha-amylase, Aspergillus niger acid stablealpha-amylase, Aspergillus niger or Aspergillus awamori glucoamylase(glaA), Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease,Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulansacetamidase, Fusarium oxysporum trypsin-like protease (as described inU.S. Pat. No. 4,288,627, which is incorporated herein by reference), andhybrids thereof. Particularly preferred promoters for use in filamentousfungal host cells are the TAKA amylase, NA2-tpi (a hybrid of thepromoters from the genes encoding Aspergillus niger neutral a amylaseand Aspergillus oryzae triose phosphate isomerase), and glaA promoters.In a yeast host, useful promoters are obtained from the Saccharomycescerevisiae enolase (ENO-1) gene, the Saccharomyces cerevisiaegalactokinase gene (GAL1), the Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase genes (ADH2/GAP),and the Saccharomyces cerevisiae 3-phosphoglycerate kinase gene. Otheruseful promoters for yeast host cells are described by Romanos et al.,1992, Yeast 8:423-488.

The nucleic acid sequence encoding said peptides and peptide conjugatesmay also be operably connected to a suitable terminator, such as thehuman growth hormone terminator (Palmiter et al., op. cit.) Preferredterminators for filamentous fungal host cells are obtained from thegenes encoding Aspergillus oryzae TAKA amylase, Aspergillus nigerglucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillusniger alpha-glucosidase, and Fusarium oxysporum trypsin-like protease.Preferred terminators for yeast host cells are obtained from the genesencoding Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C (CYC1), or Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are described by Romanos et al., 1992, supra.

The vector may further comprise elements such as polyadenylation signals(e.g., from SV 40 or the adenovirus 5 Elb region), transcriptionalenhancer sequences (e.g., the SV 40 enhancer) and translational enhancersequences (e.g., the ones encoding adenovirus VA RNAs). Furthermore,preferred polyadenylation sequences for filamentous fungal host cellsare obtained from the genes encoding Aspergillus oryzae TAKA amylase,Aspergillus niger glucoamylase, Aspergillus nidulans anthranilatesynthase, and Aspergillus niger alpha-glucosidase. Usefulpolyadenylation sequences for yeast host cells are described by Guo andSherman, 1995, Molecular Cellular Biology 15:5983-5990.

The recombinant expression vector may further comprise a DNA sequenceenabling the vector to replicate in the host cell in question. Examplesof such a sequence (when the host cell is a mammalian cell) is the SV 40or polyoma origin of replication. Examples of bacterial origins ofreplication are the origins of replication of plasmids pBR322, pUC19,pACYC177, pACYC184, pUB110, pE194, pTA1060, and pAMB1. Examples oforigin of replications for use in a yeast host cell are the 2 micronorigin of replication, the combination of CEN6 and ARS4, and thecombination of CEN3 and ARS1. The origin of replication may be onehaving a mutation to make its function temperature-sensitive in the hostcell (see, e.g., Ehrlich, 1978, Proc. Natl. Acad. Sci. USA 75:1433).

The vector may also comprise a selectable marker, e.g., a gene theproduct of which complements a defect in the host cell, such as the genecoding for dihydrofolate reductase (DHFR) or one which confersresistance to a drug, e.g., neomycin, geneticin, ampicillin, orhygromycin. Suitable markers for yeast host cells are ADE2, HIS3, LEU2,LYS2, MET3, TRP1, and URA3. A selectable marker for use in a filamentousfungal host cell may be selected from the group including, but notlimited to, amdS (acetamidase), argB (ornithine carbamoyltransferase),bar (phosphinothricin acetyltransferase), hygB (hygromycinphosphotransferase), niaD (nitrate reductase), pyrG(orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase),trpC (anthranilate synthase), and glufosinate resistance markers, aswell as equivalents from other species. Preferred for use in anAspergillus cell are the amdS and pyrG markers of Aspergillus nidulansor Aspergillus oryzae and the bar marker of Streptomyces hygroscopicus.Furthermore, selection may be accomplished by cotransformation, e.g., asdescribed in WO 91/17243, where the selectable marker is on a separatevector.

The procedures used to ligate the nucleic acid sequences coding for thepeptides and peptide conjugates, the promoter and the terminator,respectively, and to insert them into suitable vectors containing theinformation necessary for replication, are well known to persons skilledin the art (cf., for instance, Sambrook et al., op.cit.).

The host cell into which the expression vector is introduced may be anycell which is capable of producing the peptides and peptide conjugatesand is may be a eukaryotic cell, such as invertebrate (insect) cells orvertebrate cells, e.g., Xenopus laevis oocytes or mammalian cells, inparticular insect and mammalian cells. Examples of suitable mammaliancell lines are the COS (e.g., ATCC CRL 1650), BHK (e.g., ATCC CRL 1632,ATCC CCL 10) or CHO (e.g., ATCC CCL 61) cell lines. Methods fortransfecting mammalian cells and expressing DNA sequences introduced inthe cells are described in e.g., Kaufman and Sharp, 1982, J. Mol. Biol.159:601-621; Southern and Berg, 1982, J. Mol. Appl. Genet. 1:327-341;Loyter et al., 1982, Proc. Natl. Acad. Sci. USA 79:422-426; Wigler etal., 1978, Cell 14:725; Corsaro and Pearson, 1981, Somatic Cell Genetics7:603, Graham and van der Eb, 1973, Virology 52:456; Fraley et al.,1980, JBC 225:10431; Capecchi, 1980, Cell 22:479; Wiberg et al.,1983,NAR 11:7287; and Neumann et al., 1982, EMBO J. 1:841-845. The hostcell may also be a unicellular pathogen, e.g., a prokaryote, or anon-unicellular pathogen, e.g., a eukaryote. Useful unicellular cellsare bacterial cells such as gram positive bacteria including, but notlimited to, a Bacillus cell, e.g., Bacillus alkalophilus, Bacillusamyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacilluscoagulans, Bacillus lautus, Bacillus lentus, Bacillus licheniformis,Bacillus megaterium, Bacillus stearothermophilus, Bacillus subtilis, andBacillus thuringiensis; or a Streptomyces cell, e.g., Streptomyceslividans or Streptomyces murinus, or gram negative bacteria such as E.coli and Pseudomonas sp. In a preferred embodiment, the bacterial hostcell is a Bacillus lentus, Bacillus licheniformis, Bacillusstearothermophilus or Bacillus subtilis cell. The transformation of abacterial host cell may, for instance, be effected by protoplasttransformation (see, e.g., Chang and Cohen, 1979, Molecular GeneralGenetics 168:111-115), by using competent cells (see, e.g., Young andSpizizin, 1961, Journal of Bacteriology 81:823-829, or Dubnar andDavidoff Abelson, 1971, Journal of Molecular Biology 56:209-221), byelectroporation (see, e.g., Shigekawa and Dower, 1988, Biotechniques6:742-751), or by conjugation (see, e.g., Koehler and Thorne, 1987,Journal of Bacteriology 169:5771-5278). The host cell may be a fungalcell. The fungal host cell may also be a yeast cell. “Yeast” as usedherein includes ascosporogenous yeast (Endomycetales),basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti(Blastomycetes).

The medium used to culture the cells may be any conventional mediumsuitable for growing mammalian cells, such as a serum-containing orserum-free medium containing appropriate supplements, or a suitablemedium for growing insect, yeast or fungal cells. Suitable media areavailable from commercial suppliers or may be prepared according topublished recipes (e.g., in catalogues of the American Type CultureCollection).

Thus, the invention also relates to a method for producing the exendinvariants and peptide conjugates of the invention having a naturalpolypeptide sequence, comprising

-   -   a) introducing a nucleic acid sequence encoding a polypeptide        sequence comprising the peptide sequence of the exendin variant        or the peptide conjugate of the invention and a selectable        marker contained within a nucleic acid construct or a vector        into a host cell to obtain a recombinant host cell; p1 b)        selecting said recombinant host cell;    -   c) culturing said recombinant host cells under conditions        permitting the production of said polypeptide sequence;    -   d) isolating said polypeptide sequence from the culture; and    -   e) optionally cleaving said polypeptide sequence using an        appropriate protease to obtain said peptide conjugate.

The variants and peptide conjugates of the invention having a naturalpolypeptide sequence thus produced by the cells may then be recoveredfrom the culture medium by conventional procedures including separatingthe host cells from the medium by centrifugation or filtration,precipitating the proteinaceous components of the supernatant orfiltrate by means of a salt, e.g., ammonium sulphate, purification by avariety of chromatographic procedures, e.g., ion exchangechromatography, affinity chromatography, or the like. The lipophilicsubstituent(s) may be attached to the peptide of the present inventionusing procedures known in the art. In one embodiment, the lipophilicsubstituent may be attached by incorporating an amino acid with thelipophilic substituent already attached in the standard synthesis method(see, for example, synthesis of compound 7 in the Examples section).Alternatively, the substituent may be attached after the peptide hasbeen synthesized and isolated as, for example, described in WO98/08871.

The invention is further illustrated by the following examples.

EXAMPLES Peptide Synthesis, General Procedures

Apparatus and Synthetic Strategy

Peptides are synthesized batchwise in a polyethylene vessel equippedwith a polypropylene filter for filtration using9-fluorenylmethyloxycarbonyl (Fmoc) as the N-α-amino protecting groupand suitable common protection groups for side-chain functionalities(Dryland et al., 1986, J. Chem. Soc., Perkin Trans. 1:125-137).

Solvents

Solvent DMF (N,N-dimethylformamide, Riedel de-Häen, Germany) is purifiedby passing it through a column packed with a strong cation exchangeresin (Lewatit S 100 MB/H strong acid, Bayer AG Leverkusen, Germany) andanalysed for free amines prior to use by addition of3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine (Dhbt-OH) giving rise toa yellow color (Dhbt-O-anion) if free amines are present. Solvent DCM(dichloromethane, analytical grade, Riedel de-Häen, Germany) is useddirectly without purification. THF (tetrahydrofuran, analytical grade,Riedel de-Haen, Germany) is used directly without further purification.

Amino Acids

Fmoc-protected amino acids are purchased from MilliGen (UK) and fromPerSeptive Biosystems GmbH Hamburg, Germany in suitable side-chainprotected forms. FmocLys(palmitoyl)-OH is purchased from Bachem(Switzerland).

Linker

(4-hydroxymethylphenoxy)acetic acid (HMPA), Novabiochem, Switzerland iscoupled to the resin either as a preformed or in situ generated1-hydroxybenzotriazole (HObt) ester by means of DIC.

Coupling Reagents

Coupling reagent diisopropylcarbodiimide (DIC) is purchased from (Riedelde-Häen, Germany) and distilled prior to use, dicyclohexylcarbodiimide(DCC) is purchased from Merck-Schuchardt, München, Germany, and purifiedby distillation.

Solid Supports

Peptides synthesized according to the Fmoc-strategy are synthesized onthe following types of solid support using 0.05 M or higherconcentrations of Fmoc-protected activated amino acid in DMF. TentaGel Sresins 0.22-0.31 mmol/g (TentaGel S-Ram, TentaGel S RAM-Lys(Boc)Fmoc;Rapp polymere, Germany).

Catalysts and Other Reagents

Diisopropylethylamine (DIEA) is purchased from Aldrich, Germany, andethylenediamine from Fluka, piperidine and pyridine from Riedel-de Häen,Frankfurt, Germany. 4-(N,N-di-methylamino)pyridine (DMAP) is purchasedfrom Fluka, Switzerland and used as a catalyst in coupling reactionsinvolving symmetrical anhydrides. Ethanedithiol is purchased fromRiedel-de Häen, Frankfurt, Germany.3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine (Dhbt-OH) and1-hydroxybenzotriazole (HObt) are obtained from Fluka, Switzerland.

Coupling Procedures

The first amino acid is coupled as a symmetrical anhydride in DMFgenerated from the appropriate N-α-protected amino acid by means of DICor DCC. The following amino acids are coupled as preformed HObt estersmade from appropriate N-α-protected amino acids and HObt by means of DICin DMF. Acylations are checked by the ninhydrin test performed at 80° C.in order to prevent Fmoc deprotection during the test (Larsen, B. D. andHolm, A., 1994, Int. J. Peptide Protein Res. 43:1-9).

Coupling as HObt-Ester

Method a. 3 eq. N-α-amino protected amino acid is dissolved in DMFtogether with 3 eq. HObt and 3 eq DIC. The solution is left at r.t. for10 minutes and then added to the resin, which had been washed with asolution of 0.2% Dhbt-OH in DMF prior to the addition of thepreactivated amino acid.

Method b. 3 eq. N-α-amino protected amino acid is dissolved in DMFtogether with 3 eq. HObt. 3 eq DIC are added just prior to use. Thefinal solution is added to the resin.

Preformed Symmetrical Anhydride

6 eq. N-α-amino protected amino acid is dissolved in DCM and cooled to0° C. DCC or DIC (3 eq.) is added and the reaction continued for 10 min.The solvent is removed in vacuo and the residue dissolved in DMF. TheDMF-solution is filtered in case of using DCC and immediately added tothe resin followed by 0.1 eq. of DMAP.

Deprotection of the N-α-Amino Fmoc Protecting Group

Deprotection of the Fmoc group is performed by treatment with 20%piperidine in DMF (1×5 and 1×10 min.), followed by wash with DMF untilno yellow colour (Dhbt-O—) could be detected after addition of Dhbt-OHto the drained DMF.

Cleavage of Peptide from Resin with Acid

Method a. Peptides are cleaved from the resins by treatment with 95%trifluoroacetic acid (TFA, Riedel-de Häen, Frankfurt, Germany)-water v/vor with 95% TFA and 5% ethanedithiol v/v at r.t. for 2 h. The filteredresins are washed with 95% TFA-water and filtrates and washings arediluted by adding 10% acetic acid. The resulting mixture is extracted 3times with ether and finally freeze dried. The crude freeze driedproduct is analysed by high-performance liquid chromatography (HPLC) andidentified by mass spectrometry (MS).

Batchwise Peptide Synthesis on TentaGel S-RAM

TentaGel S-RAM resin (100-1000 mg, 0.22-0.31 mmol/g) is placed in apolyethylene vessel equipped with a polypropylene filter for filtration.The resin is swelled in DMF (5-10 ml), and the Fmoc group is removedaccording to the procedure described above. The following amino acidsaccording to the sequence are coupled as Fmoc-protected HObt esters (3eq.) generated in situ by means of DIC as described above. The couplingsare continued for 3 h, unless otherwise specified. The resin is drainedand washed with DMF (4×5-10 ml, 2 min each) in order to remove excessreagent. All acylations are checked by the ninhydrin test performed at80° C. After completion of the synthesis, the peptide-resin is washedwith DMF (3×5-10 ml, 5 min each), DCM (3×5-10 ml, 1 min each) andfinally diethyl ether (3×5-10 ml, 1 min each) and dried in vacuo.

HPLC Conditions

Isocratic HPLC analysis is preformed on a Shimadzu system consisting ofan LC-6A pump, an MERCK HITACHI L-4000 UV detector operated at 215 nmand a Rheodyne 7125 injection valve with a 20 μl loop. The column usedfor isocratic analysis is a Spherisorb ODS-2 (100×3 mm; 5-μm particles)(MicroLab, Aarhus, Denmark). HPLC analysis using gradients is performedon a MERCK-HITACHI L-6200 Intelligent pump, an MERCK HITACHI L-4000 UVdetector operated at 215 nm and a Rheodyne 7125 injection valve with a20 μl loop, or on a Waters 600 E instrument equipped with a Waters 996photodiode array detector. The columns used are a Rescorce™ RPC 1 ml(Waters) or a LiChroCART 125-4, LiChrospher 100 RP-18 (5 μm) (Merck).

Buffer A is 0.1 vol % TFA in water and buffer B 90 vol % acetonitrile,9.9 vol % water and 0.1 vol % TFA. The buffers are pumped through thecolumns at a flow rate of 1.3-1.5 ml/min using either of the followinggradients for peptide analysis 1) Linear gradient from 0% -100% B (30min) or 2) 0% B (2 min) linear gradient from 0-50% B (23 min) 50-100% B(5 min).

For Preparative HPLC, purification is performed on a Waters 600 Einstrument equipped with a Waters 996 photodiode array detector. Thecolumn used is a Waters Delta-Pak C-18 15 μm, 100 Å, 25×100 mm. Gradient“2)” is used with a flow rate of 9 ml/min.

Mass Spectroscopy

Mass spectra are obtained on a Finnigan Mat LCQ instrument equipped withan electrospray (ESI) probe (ES-MS) and on a TofSpec E, FisonsInstrument (MALDI-TOF) using β-cyano-p-hydroxycinnamic acid as matrix.Alternatively, spectra may be obtained by a Micromass LCT instrument.

Peptide Synthesis of Prior Art Peptides

(i) Peptide synthesis of Compound (i),H-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH₂ (exendin-4(1-39)-NH₂) (SEQ IDNO:102) on TentaGel S-RAM.

Dry TentaGel S-RAM resin (0.25 mmol/g, 1000 mg) is placed in apolyethylene vessel equipped with a polypropylene filter for filtrationand swelled for two hours in DMF (5 ml). The Fmoc group is removedaccording to the procedure described above, and the peptide according tothe sequence is assembled as described under “Batchwise peptidesynthesis on TentaGel S-RAM resins”. After completion of the synthesis,the peptide-resin is washed with DMF (3×5 ml, 1 min each), DCM (3×5 ml,1 min each), diethyl ether (3×5 ml, 1 min each) and dried in vacuo. Thepeptide is cleaved from the resin according to method a as describedabove and freeze dried from acetic acid. The crude peptide is purifiedby preparative HPLC using the procedure described above. The purifiedproduct is found to be homogeneous and the purity is found to be betterthan 90%. The identity of the peptide is confirmed by ES-MS. Yield 17%.

(ii) Peptide synthesis of Compound (ii),H-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-NH₂ (SEQ ID NO: 129)

(des Ser³⁹ exendin-4(1-39)-NH₂) (SEQ ID NO: 129) on TentaGel S-RAM.

Dry TentaGel S-RAM resin (0.25 mmol/g, 1000 mg) is placed in apolyethylene vessel equipped with a polypropylene filter for filtrationand swelled for two hours in DMF (5 ml). The Fmoc group is removedaccording to the procedure described above, and the peptide according tothe sequence is assembled as described under “Batchwise peptidesynthesis on TentaGel S-RAM resins”. After completion of the synthesis,the peptide-resin is washed with DMF (3×5 ml, 1 min each), DCM (3×5 ml,1 min each), diethyl ether (3×5 ml, 1 min each) and dried in vacuo. Thepeptide is cleaved from the resin according to method a as describedabove and freeze dried from acetic acid. The crude peptide is purifiedby preparative HPLC using the procedure described above. The purifiedproduct is found to be homogeneous and the purity is found to be betterthan 97%. The identity of the peptide is confirmed by ES-MS. Yield 22%.

(iii) Peptide synthesis of Compound (iii),H-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-NH₂(SEQ ID NO: 87)

(Gly⁸-GLP1-(7-36)(Human)-NH₂) (SEQ ID NO:87) on TentaGel S-RAM.

Dry TentaGel S-RAM resin (0.25 mmol/g, 1000 mg) is placed in apolyethylene vessel equipped with a polypropylene filter for filtrationand swelled for two hours in DMF (5 ml). The Fmoc group is removedaccording to the procedure described above, and the peptide according tothe sequence is assembled as described under “Batchwise peptidesynthesis on TentaGel S-RAM resins”. After completion of the synthesis,the peptide-resin is washed with DMF (3×5 ml, 1 min each), DCM (3×5 ml,1 min each), diethyl ether (3×5 ml, 1 min each) and dried in vacuo. Thepeptide is cleaved from the resin according to method a as describedabove and freeze dried from acetic acid. The crude peptide is purifiedby preparative HPLC using the procedure described above. The purifiedproduct is found to be homogeneous and the purity is found to be betterthan 95%. The identity of the peptide is confirmed by ES-MS. Yield 9%.

Synthesis of Peptide Sequences of the Invention

1. Peptide synthesis of Compound 1,H-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Ser-NH₂ (SEQ ID NO: 101)

(des Pro³⁶-exendin-4(1-39)-NH₂) (SEQ ID NO:101) on TentaGel S-RAM.

Dry TentaGel S-RAM resin (0.25 mmol/g, 1500 mg) is placed in apolyethylene vessel equipped with a polypropylene filter for filtrationand swelled for two hours in DMF (5 ml) The Fmoc group is removedaccording to the procedure described above, and the peptide according tothe sequence is assembled as described under “Batchwise peptidesynthesis on TentaGel S-RAM resins”. After completion of the synthesis,the peptide-resin is washed with DMF (3×5 ml, 1 min each), DCM (3×5 ml,1 min each), diethyl ether (3×5 ml, 1 min each) and dried in vacuo. Thepeptide is cleaved from the resin according to method a as describedabove and freeze dried from acetic acid. The crude peptide is purifiedby preparative HPLC using the procedure described above. The purifiedproduct is found to be homogeneous and the purity is found to be betterthan 95%. The identity of the peptide is confirmed by ES-MS. Yield18.3%.

2. Peptide synthesis of Compound 2,H-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Ser-(Lys)₆-NH₂ (SEQ ID NO: 93)

(des-Pro³⁶-exendin-4(1-39)-Lys₆-NH₂) (SEQ ID NO:93) on TentaGelS-RAM-Lys(Boc)Fmoc.

Dry TentaGel S-RAM-Lys(Boc)Fmoc resin (0.22 mmol/g, 1500 mg) is placedin a poly-ethylene vessel equipped with a polypropylene filter forfiltration and swelled for two hours in DMF (5 ml). The Fmoc group onthe first lysine is removed as described above and the synthesis iscontinued until finishing the peptide sequence as described under“Batchwise peptide synthesis on TentaGel S-Ram-Lys(Boc)Fmoc”. Aftercompletion of the synthesis, the peptide-resin is washed with DMF (3×5ml, 1 min each), DCM (3×5 ml, 1 min each), diethyl ether (3×5 ml, 1 mineach) and dried in vacuo. The peptide is cleaved from the resinaccording to method a as described above and freeze dried from aceticacid. The crude freeze dried product is purified by preparative HPLCusing the procedure described above. The purified product is found to behomogeneous and the purity is found to be better than 95%. The identityof the peptide is confirmed by ES-MS. Yield 22.1%.

3. Peptide synthesis of Compound 3,H-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-(Lys)₆-NH₂ (SEQ ID NO: 92)

(exendin-4(1-39)-Lys6-NH₂) (SEQ ID NO:92) on TentaGelS-RAM-Lys(Boc)Fmoc.

Dry TentaGel S-RAM-Lys(Boc)Fmoc resin (0.22 mmol/g, 1000 mg) is placedin a poly-ethylene vessel equipped with a polypropylene filter forfiltration and swelled for two hours in DMF (5 ml). The Fmoc group onthe first lysine is removed as described above and the synthesis iscontinued until finishing the peptide sequence as described under“Batchwise peptide synthesis on TentaGel S-Ram-Lys(Boc)Fmoc”. Aftercompletion of the synthesis, the peptide-resin is washed with DMF (3×5ml, 1 min each), DCM (3×5 ml, 1 min each), diethyl ether (3×5 ml, 1 mineach) and dried in vacuo. The peptide is cleaved from the resinaccording to method a as described above and freeze dried from aceticacid. The crude freeze dried product is purified by preparative HPLCusing the procedure described above. The purified product is found to behomogeneous and the purity is found to be better than 90%. The identityof the peptide is confirmed by ES-MS. Yield 20.5%.

4. Peptide synthesis of Compound 4,H-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-(Lys)₆-NH₂(SEQ ID NO: 88) (Gly⁸-GLP1-(7-36)(Human)-Lys₆-NH₂) (SEQ ID NO:88) onTentaGel S-RAM-Lys(Boc)Fmoc.

Dry TentaGel S-RAM-Lys(Boc)Fmoc resin (0.22 mmol/g, 1000 mg) is placedin a polyethylene vessel equipped with a polypropylene filter forfiltration and swelled for two hours in DMF (5 ml). The Fmoc group onthe first lysine is removed as described above and the synthesis iscontinued until finishing the peptide sequence as described under“Batchwise peptide synthesis on TentaGel S-Ram-Lys(Boc)Fmoc”. Aftercompletion of the synthesis, the peptide-resin is washed with DMF (3×5ml, 1 min each), DCM (3×5 ml, 1 min each), diethyl ether (3×5 ml, 1 mineach) and dried in vacuo. The peptide is cleaved from the resinaccording to method a as described above and freeze dried from aceticacid. The crude freeze dried product is purified by preparative HPLCusing the procedure described above. The purified product is found to behomogeneous and the purity is found to be better than 95%. The identityof the peptide is confirmed by ES-MS. Yield 11.7%.

4a. Peptide synthesis of Compound 4,H-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Lys-Lys-Lys-Lys-Lys-Lys-NH₂ (SEQ ID NO: 88)

([Gly⁸]hGLP-1(7-36)-(Lys)₆-NH₂) (SEQ ID NO:88) on TentaGelS-RAM-Lys(Boc)Fmoc.

Dry TentaGel S-RAM-Lys(Boc)Fmoc resin (0.22 mmol/g, 2013 mg) is placedin a glass vessel equipped with a polypropylene filter for filtrationand swelled for two hours in DMF (5 ml). The Fmoc group on the firstlysine is removed as described above and the synthesis is continueduntil finishing the peptide sequence as described under “Batchwisepeptide synthesis on TentaGel S-Ram-Lys(Boc)Fmoc”. After completion ofthe synthesis, the peptide-resin is washed with DMF (3×5 ml, 1 mineach), DCM (3×5 ml, 1 min each), diethyl ether (3×5 ml, I min each) anddried in vacuo. The peptide is cleaved from the resin according tomethod a as described above and freeze dried from acetic acid. The crudefreeze dried product is purified by preparative HPLC using the proceduredescribed above. The purified product is found to be homogeneous and thepurity is found to be better than 90%. The identity of the peptide isconfirmed by ES-MS. Yield 13%.

5. Peptide synthesis of Compound 5,H-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Lys(palmitoyl)-(Lys)₆-NH₂ (SEQ ID NO: 89)

([Gly⁸, Lys³⁷(palmitoyl)]GLP1-(7-36)(Human)-(Lys)7-NH₂) (SEQ ID NO:89)on TentaGel S-RAM-Lys(Boc)Fmoc.

Dry TentaGel S-RAM-Lys(Boc)Fmoc resin (0.22 mmol/g, 1000 mg) is placedin a polyethylene vessel equipped with a polypropylene filter forfiltration and swelled for two hours in DMF (5 ml). The Fmoc group onthe first lysine is removed as described above and the synthesis iscontinued until finishing the peptide sequence as described under“Batchwise peptide synthesis on TentaGel S-Ram-Lys(Boc)Fmoc”. Thereagent Fmoc-Lys(palmitoyl)-OH is coupled in a slightly modified mannerdue to its poor solubility in DMF.

Approximately 400 mg of Fmoc-Lys(palmitoyl)-OH is dissolved inapproximately 6 ml THF rather than DMF. After completion of thesynthesis, the peptide-resin is washed with DMF (3×5 ml, 1 min each),DCM (3×5 ml, 1 min each), diethyl ether (3×5 ml, 1 min each) and driedin vacuo. The peptide is cleaved from the resin according to method b asdescribed above and freeze dried from acetic acid. The crude freezedried product is purified by preparative HPLC using the proceduredescribed above. The purified product is found to be homogeneous and thepurity is found to be better than 95%. The identity of the peptide isconfirmed by ES-MS. Yield 9.3%.

6. Peptide synthesis of Compound 6,H-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys(palmitoyl)-Gly-Arg-(Lys)₆-NH₂(SEQ ID NO: 90)

([Gly⁸. Lys³⁴(palmitoyl)]GLP1-(7-36)(Human)-(Lys)6-NH₂) (SEQ ID NO:90)on TentaGel S-RAM-Lys(Boc)Fmoc.

Dry TentaGel S-RAM-Lys(Boc)Fmoc resin (0.22 mmol/g, 1000 mg) is placedin a polyethylene vessel equipped with a polypropylene filter forfiltration and swelled for two hours in DMF (5 ml). The Fmoc group onthe first lysine is removed as described above and the synthesis iscontinued until finishing the peptide sequence as described under“Batchwise peptide synthesis on TentaGel S-Ram-Lys(Boc)Fmoc”. Thereagent Fmoc-Lys(palmitoyl)-OH is coupled in a slightly modified mannerdue to its poor solubility in DMF.

Approximately 400 mg of Fmoc-Lys(palmitoyl)-OH is dissolved inapproximately 6 ml THF rather than DMF. After completion of thesynthesis, the peptide-resin is washed with DMF (3×5 ml, 1 min each),DCM (3×5 ml, 1 min each), diethyl ether (3×5 ml, 1 min each) and driedin vacuo. The peptide is cleaved from the resin according to method a asdescribed above and freeze dried from acetic acid. The crude freezedried product is purified by preparative HPLC using the proceduredescribed above. The purified product is found to be homogeneous and thepurity is found to be better than 90%. The identity of the peptide isconfirmed by ES-MS. Yield 4.2%.

7. Peptide synthesis of Compound 7,H-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(palmitoyl)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg- (Lys)₆-NH₂ (SEQID NO: 103)

([Gly⁸, Lys²⁶(palmitoyl)]GLP1-(7-36)(Human)-(Lys)6-NH₂) (SEQ ID NO:103)on TentaGel S-RAM-Lys(Boc)Fmoc.

Dry TentaGel S-RAM-Lys(Boc)Fmoc resin (0.22 mmol/g, 1000 mg) is placedin a polyethylene vessel equipped with a polypropylene filter forfiltration and swelled for two hours in DMF (5 ml). The Fmoc group onthe first lysine is removed as described above and the synthesis iscontinued until finishing the peptide sequence as described under“Batchwise peptide synthesis on TentaGel S-Ram-Lys(Boc)Fmoc”. Thereagent Fmoc-Lys(palmitoyl)-OH is coupled in a slightly modified mannerdue to its poor solubility in DMF.

Approximately 400 mg of Fmoc-Lys(palmitoyl)-OH is dissolved inapproximately 6 ml THF rather than DMF. After completion of thesynthesis, the peptide-resin is washed with DMF (3×5 ml, 1 min each),DCM (3×5 ml, 1 min each), diethyl ether (3×5 ml, 1 min each) and driedin vacuo. The peptide is cleaved from the resin according to method a asdescribed above and freeze dried from acetic acid. The crude freezedried product is purified by preparative HPLC using the proceduredescribed above. The purified product is found to be homogeneous and thepurity is found to be better than 90%. The identity of the peptide isconfirmed by ES-MS. Yield 2.2%.

8. Peptide synthesis of Compound 8,H-Lys-Lys-Lys-Lys-Lys-Lys-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Ser-NH₂ (SEQ ID NO: 149)

(H-(Lys)₆-des Pro³⁶exendin-4(1-39)-NH₂) (SEQ ID NO: 149) on TentaGelS-RAM-Fmoc.

Dry TentaGel S-RAM-Fmoc resin (0.23 mmol/g, 1000 mg) is placed in apoly-ethylene vessel equipped with a polypropylene filter for filtrationand swelled for two hours in DMF (5 ml). The Fmoc group on the resin isremoved as described above and the synthesis is continued untilfinishing the peptide sequence as described under “Batchwise peptidesynthesis on TentaGel S-Ram-Fmoc”. After completion of the synthesis,the peptide-resin is washed with DMF (3×5 ml, 1 min each), DCM (3×5 ml,1 min each), diethyl ether (3×5 ml, 1 min each) and dried in vacuo. Thepeptide is cleaved from the resin according to method a as describedabove and freeze dried from acetic acid. The crude freeze dried productis purified by preparative HPLC using the procedure described above. Thepurified product is found to be homogeneous and the purity is found tobe better than 95%. The identity of the peptide is confirmed by ES-MS.Yield 26%.

9. Peptide synthesis of Compound 9,H-Lys₆-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Ser-(Lys)₆-NH₂ (SEQ ID NO: 150)

(H-Lys₆-des Pro³⁶exendin-4(1-39)-Lys₆-NH₂) (SEQ ID NO: 150) on TentaGelS-RAM-Lys(Boc)Fmoc.

Dry TentaGel S-RAM-Lys(Boc)Fmoc resin (0.22 mmol/g, 1000 mg) is placedin a poly-ethylene vessel equipped with a polypropylene filter forfiltration and swelled for two hours in DMF (5 ml). The Fmoc group onthe first lysine is removed as described above and the synthesis iscontinued until finishing the peptide sequence as described under“Batchwise peptide synthesis on TentaGel S-Ram-Lys(Boc)Fmoc”. Aftercompletion of the synthesis, the peptide-resin is washed with DMF (3×5ml, 1 min each), DCM (3×5 ml, 1 min each), diethyl ether (3×5 ml, 1 mineach) and dried in vacuo. The peptide is cleaved from the resinaccording to method a as described above and freeze dried from aceticacid. The crude freeze dried product is purified by preparative HPLCusing the procedure described above. The purified product is found to behomogeneous and the purity is found to be better than 90%. The identityof the peptide is confirmed by ES-MS. Yield 32%.

10. Peptide synthesis of Compound 10,H-Lys-Lys-Lys-Lys-Lys-Lys-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Lys-Lys-Lys-Lys-Lys-Lys-NH₂ (SEQ ID NO: 118)(H-(Lys)₆-([Gly⁸]hGLP-1(7-36)-(Lys)₆-NH₂) (SEQ ID NO: 118) on TentaGelS-RAM-Lys(Boc)Fmoc.

Dry TentaGel S-RAM-Lys(Boc)Fmoc resin (022 mmol/g, 1000 mg) is placed ina poly-ethylene vessel equipped with a polypropylene filter forfiltration and swelled for two hours in DMF (5 ml). The Fmoc group onthe first lysine is removed as described above and the synthesis iscontinued until finishing the peptide sequence as described under“Batchwise peptide synthesis on TentaGel S-Ram-Lys(Boc)Fmoc”. Aftercompletion of the synthesis, the peptide-resin is washed with DMF (3×5ml, 1 min each), DCM (3×5 ml, 1 min each), diethyl ether (3×5 ml, 1 mineach) and dried in vacuo. The peptide is cleaved from the resinaccording to method a as described above and freeze dried from aceticacid. The crude freeze dried product is purified by preparative HPLCusing the procedure described above. The purified product is found to behomogeneous and the purity is found to be better than 90%. The identityof the peptide is confirmed by ES-MS. Yield 18%.

11. Peptide synthesis of Compound 11,H-Lys-Lys-Lys-Lys-Lys-Lys-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-NH₂ (SEQ ID NO: 119)

(H-(Lys)₆-[Gly⁸]hGLP-1(7-36)-NH₂) (SEQ ID NO: 119) on TentaGelS-RAM-Lys(Boc)Fmoc.

Dry TentaGel S-RAM-Fmoc resin (0.23 mmol/g, 1000 mg) is placed in apoly-ethylene vessel equipped with a polypropylene filter for filtrationand swelled for two hours in DMF (5 ml). The Fmoc group on the resin isremoved as described above and the synthesis is continued untilfinishing the peptide sequence as described under “Batchwise peptidesynthesis on TentaGel S-Ram-Fmoc”. After completion of the synthesis,the peptide-resin is washed with DMF (3×5 ml, 1 min each), DCM (3×5 ml,1 min each), diethyl ether (3×5 nil, 1 min each) and dried in vacuo. Thepeptide is cleaved from the resin according to method a as describedabove and freeze dried from acetic acid. The crude freeze dried productis purified by preparative HPLC using the procedure described above. Thepurified product is found to be homogeneous and the purity is found tobe better than 98%. The identity of the peptide is confirmed by ES-MS.Yield 15%.

12. Peptide synthesis of Compound 12,H-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Lys-Lys-Lys-Lys-Lys-Lys-Lys-Lys-NH₂ (SEQ ID NO: 120)

([Gly⁸]hGLP-1(7-36)-(Lys)₈-NH₂) (SEQ ID NO: 120) on TentaGelS-RAM-Lys(Boc)Fmoc.

Dry TentaGel S-RAM-Lys(Boc)Fmoc resin (0.22 mmol/g, 1000 mg) is placedin a poly-ethylene vessel equipped with a polypropylene filter forfiltration and swelled for two hours in DMF (5 ml). The Fmoc group onthe first lysine is removed as described above and the synthesis iscontinued until finishing the peptide sequence as described under“Batchwise peptide synthesis on TentaGel S-Ram-Lys(Boc)Fmoc”. Aftercompletion of the synthesis, the peptide-resin is washed with DMF (3×5ml, 1 min each), DCM (3×5 ml, 1 min each), diethyl ether (3×5 ml, 1 mineach) and dried in vacuo. The peptide is cleaved from the resinaccording to method a as described above and freeze dried from aceticacid. The crude freeze dried product is purified by preparative HPLCusing the procedure described above. The purified product is found to behomogeneous and the purity is found to be better than 98%. The identityof the peptide is confirmed by ES-MS. Yield 4.2%.

13. Peptide synthesis of Compound 13,H-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Lys-Lys-Lys-Lys-Lys-Lys-Lys-Lys-Lys-Lys-NH₂ (SEQ ID NO: 121)

([Gly⁸]hGLP-1(7-36)-(Lys)₁₀-NH₂) (SEQ ID NO: 121) on TentaGelS-RAM-Lys(Boe)Fmoc.

Dry TentaGel S-RAM-Lys(Boc)Fmoc resin (0.22 mmol/g, 1000 mg) is placedin a poly-ethylene vessel equipped with a polypropylene filter forfiltration and swelled for two hours in DMF (5 ml). The Fmoc group onthe first lysine is removed as described above and the synthesis iscontinued until finishing the peptide sequence as described under“Batchwise peptide synthesis on TentaGel S-Ram-Lys(Boc)Fmoc”. Aftercompletion of the synthesis, the peptide-resin is washed with DMF (3×5ml, 1 min each), DCM (3×5 ml, 1 min each), diethyl ether (3×5 ml, 1 mineach) and dried in vacuo. The peptide is cleaved from the resinaccording to method a as described above and freeze dried from aceticacid. The crude freeze dried product is purified by preparative HPLCusing the procedure described above. The purified product is found to behomogeneous and the purity is found to be better than 95%. The identityof the peptide is confirmed by ES-MS. Yield 2%.

14. Peptide synthesis of Compound 14,H-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Ser-NH₂ (SEQ ID NO: 132)

(H-des Pro³⁶, Pro³⁷, Pro³⁸exendin-4(1-39)-NH₂) (SEQ ID NO: 132) onTentaGel S-RAM-Fmoc.

Dry TentaGel S-RAM-Fmoc resin (0.23 mmol/g, 1000 mg) is placed in apoly-ethylene vessel equipped with a polypropylene filter for filtrationand swelled for two hours in DMF (5 ml). The Fmoc group on the resin isremoved as described above and the synthesis is continued untilfinishing the peptide sequence as described under “Batchwise peptidesynthesis on TentaGel S-Ram-Fmoc”. After completion of the synthesis,the peptide-resin is washed with DMF (3×5 ml, 1 min each), DCM (3×5 ml,1 min each), diethyl ether (3×5 ml, 1 min each) and dried in vacuo. Thepeptide is cleaved from the resin according to method a as describedabove and freeze dried from acetic acid. The crude freeze dried productis purified by preparative HPLC using the procedure described above. Thepurified product is found to be homogeneous and the purity is found tobe better than 95%. The identity of the peptide is confirmed by ES-MS.Yield 11%.

15. Peptide synthesis of Compound 15,H-Lys-Lys-Lys-Lys-Lys-Lys-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Ser-NH₂ (SEQ ID NO: 134)(H-(Lys)₆-des Pro³⁶, Pro³⁷, Pro³⁸exendin-4(1-39)-NH₂) (SEQ ID NO: 134)on TentaGel S-RAM-Fmoc.

Dry TentaGel S-RAM-Fmoc resin (0.23 mmol/g, 1000 mg) is placed in apoly-ethylene vessel equipped with a polypropylene filter for filtrationand swelled for two hours in DMF (5 ml). The Fmoc group on the resin isremoved as described above and the synthesis is continued untilfinishing the peptide sequence as described under “Batchwise peptidesynthesis on TentaGel S-Ram-Fmoc”. After completion of the synthesis,the peptide-resin is washed with DMF (3×5 ml, 1 min each), DCM (3×5 ml,1 min each), diethyl ether (3×5 ml, 1 min each) and dried in vacuo. Thepeptide is cleaved from the resin according to method a as describedabove and freeze dried from acetic acid. The crude freeze dried productis purified by preparative HPLC using the procedure described above. Thepurified product is found to be homogeneous and the purity is found tobe better than 94%. The identity of the peptide is confirmed by ES-MS.Yield 17%.

16. Peptide synthesis of compound 16,H-Asn-Glu-Glu-Glu-Glu-Glu-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Ser-NH₂ (SEQ ID NO: 137)(H-Asn-(Glu)₅-des Pro³⁶, Pro³⁷, Pro³⁸exendin-4(1-39)-NH²) (SEQ ID NO:137) on TentaGel S-RAM-Fmoc.

Dry TentaGel S-RAM-Fmoc resin (0.23 mmol/g, 1000 mg) is placed in apoly-ethylene vessel equipped with a polypropylene filter for filtrationand swelled for two hours in DMF (5 ml). The Fmoc group on the resin isremoved as described above and the synthesis is continued untilfinishing the peptide sequence as described under “Batchwise peptidesynthesis on TentaGel S-Ram-Fmoc”. After completion of the synthesis,the peptide-resin is washed with DMF (3×5 ml, 1 min each), DCM (3×5 ml,1 min each), diethyl ether (3×5 ml, 1 min each) and dried in vacuo. Thepeptide is cleaved from the resin according to method a as describedabove and freeze dried from acetic acid. The crude freeze dried productis purified by preparative HPLC using the procedure described above. Thepurified product is found to be homogeneous and the purity is found tobe better than 90%. The identity of the peptide is confirmed by ES-MS.Yield 9%.

17. Peptide synthesis of Compound 17, Compound 3,H-(Lys)₆-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Ser-(Lys)₆-NH₂ (SEQ ID NO: 135)(H-(Lys)₆-des Pro³⁶, Pro³⁷, Pro³⁸exendin-4(1-39)-(Lys)₆-NH₂) (SEQ ID NO:135) on TentaGel S-RAM-Lys(Boc)Fmoc.

Dry TentaGel S-RAM-Lys(Boc)Fmoc resin (0.22 mmol/g, 1000 mg) is placedin a poly-ethylene vessel equipped with a polypropylene filter forfiltration and swelled for two hours in DMF (5 ml). The Fmoc group onthe first lysine is removed as described above and the synthesis iscontinued until finishing the peptide sequence as described under“Batchwise peptide synthesis on TentaGel S-Ram-Lys(Boc)Fmoc”. Aftercompletion of the synthesis, the peptide-resin is washed with DMF (3×5ml, 1 min each), DCM (3×5 ml, 1 min each), diethyl ether (3×5 ml, 1 mineach) and dried in vacuo. The peptide is cleaved from the resinaccording to method a as described above and freeze dried from aceticacid. The crude freeze dried product is purified by preparative HPLCusing the procedure described above. The purified product is found to behomogeneous and the purity is found to be better than 90%. The identityof the peptide is confirmed by ES-MS. Yield 10%.

18. Peptide synthesis of Compound 18,H-Asn-Glu-Glu-Glu-Glu-Glu-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Ser-(Lys)₆-NH₂ (SEQ ID NO: 136)(H-Asn-(Glu)₅-des Pro³⁶, Pro³⁷, Pro³⁸exendin-4(1-39)-(Lys)₆-NH₂) (SEQ IDNO: 136) on TentaGel S-RAM-Lys(Boc)Fmoc.

Dry TentaGel S-RAM-Lys(Boc)Fmoc resin (0.22 mmol/g, 1000 mg) is placedin a poly-ethylene vessel equipped with a polypropylene filter forfiltration and swelled for two hours in DMF (5 ml). The Fmoc group onthe first lysine is removed as described above and the synthesis iscontinued until finishing the peptide sequence as described under“Batchwise peptide synthesis on TentaGel S-Ram-Lys(Boc)Fmoc”. Aftercompletion of the synthesis, the peptide-resin is washed with DMF (3×5ml, 1 min each), DCM (3×5 ml, 1 min each), diethyl ether (3×5 ml, 1 mineach) and dried in vacuo. The peptide is cleaved from the resinaccording to method a as described above and freeze dried from aceticacid. The crude freeze dried product is purified by preparative HPLCusing the procedure described above. The purified product is found to behomogeneous and the purity is found to be better than 92%. The identityof the peptide is confirmed by ES-MS. Yield 14%.

19. Peptide synthesis of Compound 19,H-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Ser-(Lys)₆-NH₂ (SEQ ID NO: 133)

(des Pro³⁶, Pro³⁷, Pro³⁸exendin-4(1-39)-(Lys)₆-NH₂) (SEQ ID NO: 133) onTentaGel S-RAM-Lys(Boc)Fmoc.

Dry TentaGel S-RAM-Lys(Boc)Fmoc resin (0.22 mmol/g, 1000 mg) is placedin a poly-ethylene vessel equipped with a polypropylene filter forfiltration and swelled for two hours in DMF (5 ml). The Fmoc group onthe first lysine is removed as described above and the synthesis iscontinued until finishing the peptide sequence as described under“Batchwise peptide synthesis on TentaGel S-Ram-Lys(Boc)Fmoc”. Aftercompletion of the synthesis, the peptide-resin is washed with DMF (3×5ml, 1 min each), DCM (3×5 ml, 1 min each), diethyl ether (3×5 ml, 1 mineach) and dried in vacuo. The peptide is cleaved from the resinaccording to method a as described above and freeze dried from aceticacid. The crude freeze dried product is purified by preparative HPLCusing the procedure described above. The purified product is found to behomogeneous and the purity is found to be better than 97%. The identityof the peptide is confirmed by ES-MS. Yield 19%.

20. Recombinant preparation of Compound 2

Construction of the pYES0010 Expression Vector

A synthetic cDNA was constructed for heterolog expression in yeast. Theprotein sequence encoding Compound 2 was back translated using aSaccharomyces cerevisiae codon usage table (Saccharomyces GenomeDatabase). To enable translation of the synthetic cDNA an additional ATGstart codon was added to the 5′ end and a TAA stop codon was added tothe 3′ end. The construct was inserted into HindIII and The EcoRI siteof the pYES2 shuttle vector comprising an ampicilline resistance gene,and the new construct was designated pYES0010, cf. FIG. 6. pYES0010 wassubsequently transformed into E. coli and subjected to ampicillinselection pressure. Positive clones were selected and sequenced.

Transformation into Yeast.

In order to make transform the pYES0010 into the yeast haploid INVScl:MATa his3delta1 leu2 trp1-289 ura3-52. Yeast were grown in YPD medium(1% yeast extract, 2% peptone, 2% glucose, and 0.004% adenine sulfate)at 30 C to saturation. 1ml of culture was harvested for transformations.2 μl of 10 mg/ml carrier DNA was added and 1 μg of pYES0010 was addedand mixed. 0.5 ml (45% PEG 4000, 1M Li OAc, 0.5M EDTA and 1M Tris-HCl(pH 7.5) was added and mixed. Finally 20 μl 1M DTT was added and themixture was incubated for 16 h at room temperature. After incubation thecells were heat shocked at 42 C for 10 min and plated selective plates(6.7% yeast nitrogen base, 2% glucose, 20 μg/ml adenine, 20 μg/mlarginine, 29 μg/ml isoleucine, 20 μg/ml histidine, 60 μg/ml leucine, 20μg/ml lysine, 20 μg/ml tryptophan, 20 μg/ml methionine 50 μg/mlphenylalanine 150 μg/ml valine, 30 μg/ml Tyrosine and 2.5% agar. Plateswere incubated at 30 C for 3 to 5 days until transformants appear.

Expression and Purification of Compound 2.

Transformants were cultivated in selective media (6.7% Yeast nitrogenbase, 2% glucose, 20 μg/ml adenine, 20 μg/ml arginine, 29 μg/mlisoleucine, 20 μg/ml histidine, 60 μg/ml leucine, 20 μg/ml lysine, 20μg/ml Tryptophan, 20 μg/ml methionine 50 μg/ml phenylalanine 150 μg/mlvaline, 30 μg/ml Tyrosine) for 1.5 days. The cells were harvested andresuspended in galactose induction medium (6.7% Yeast nitrogen base, 4%galactose, 20 μg/ml adenine, 20 μg/ml arginine, 29 μg/ml isoleucine, 20μg/ml histidine, 60 μg/ml leucine, 20 μg/ml lysine, 20 μg/ml Tryptophan,20 μg/ml methionine 50 μg/ml phenylalanine 150 μg/ml valine, 30 μg/mlTyrosine for 1 day. The cells were harvested and homogenized in 10 mMTris-HCl pH 7.5 containing protease inhibitors (Roche). The lysate wasclarified centrifugation at 20.000×g for 30 min. The supernatant wasloaded onto a Superdex 12 HR 10/30 column (Amersham Pharmacia Biotech)equilibrated with 10 mM Tris-HCl pH 7.5. The column was eluted in 50 Mmammonia bicarbonate buffer pH 8.0. Samples containing recombinantCompound 2 were pooled. The N-terminal methionine was removed bymethionine aminopeptidase and the samples were further purified on aHPLC Column.

HPLC Settings for Compound 2 Purification.

HPLC column : Kromasil RP C8; K 100-10-C8 nr. CER 2230. compound

Temp: 22 C

Flow rate: 35 ml/min

HPLC solvents:

A: 0.10% trifluoroacetic acid in water

B: 0.10% trifluoroacetic acid in acetonitrile: water 90:10.

Compound 2 was eluted from the HPLC column with 0.10% trifluoroaceticacid in 20% to 80% Acetonitrile in 40 min.

21. Injection Formulations of Peptide

Fixed dose formulations of peptide for intra venous injection areprepared by dissolving the peptide in sterile, isotonic saline, andstoring the resulting solution in glass ampoules filled with inert gasunder sterile conditions. Each dose of the peptide is stored dry inampoules or capped vials filled with inert gas. Multi-dose formulationsof peptide for intra venous injection are prepared by dissolving thepeptide in sterile, isotonic saline, storing the resulting solution incapped vials, if necessary adding preservative (for instance 0.1%parahydroxybenzoate, 1% benzyl alcohol or 0.1% chlorocresole).

Example of multi-dose peptide formulation:

Compound 2 12.25 mg Sodiumdihydrogenphosphate 1.380 gParahydroxybenzoate 0.1 g Aqua ad injectabile 100 ml

22. Stability Experiments

In vitro stability studies with the present peptides and peptideconjugates in the presence of selected proteolytic enzymes are appliedas a tool for evaluating the protection of said peptides againstproteolysis in vivo. The aim of the experiments performed was to measureand compare the in vitro stability of Compounds 4, 5, 6 and Ito that ofthe prior art compounds Compound(iii) H-(Gly)-hGLP-1(7-36)-NH₂ (SEQ IDNO: 87) and hGLP-1(7-36)-NH₂ (SEQ ID NO: 114) in solutions of one ormore of the enzymes leucine aminopeptidase, carboxypeptidase A anddipeptidyl aminopeptidase IV at 37° C.

Materials and Apparatus for In Vitro Stability

Water used was of highest quality obtained from a Milli-Q watertreatment system (Millipore, Bedford, Mass., USA). Acetonitrile (ACN)was of super gradient quality obtained from Labscan Ltd. (Dublin,Ireland). Trifluoracetic acid (TFA) 99.9%, dihydrogen phosphate(NaH₂PO₄), sodium hydroxide (NaOH) and all other chemicals used were ofanalytical grade. Leucine aminopeptidase (EC 3.4.11.1), CarboxypeptidaseA (EC 3.4.17.1) and Dipeptidyl peptidase (Dipeptidyl aminopeptidase IV,EC 3.4.14.5) were all obtained from Sigma (St. Louis, Mo., USA).Gradient HPLC analysis was done using a Hewlett Packard HP 1100 HPLCsystem consisting of a HP 1100 Binary Pump, a HP 1100 Autosampler, A HP1100 Column Thermostat and a HP 1100 Variable Wavelength Detector.Hewlett Packard Chemstation for LC software (Rev. A.06.01) was used forinstrument control and data acquisition. A Vydac 238TP54 (150×4.6 mmI.D.) column packed with 5 m, C18, 300 particles was used with theinstrument. A SHT200D block heater from Stuart Scientific was used forheating of the peptide/enzyme solutions during the stabilityexperiments. The degradation of the test compounds was studied at 37° C.in 50 mM phosphate buffer solutions of pH 7.4 containing leucineaminopeptidase (25 U/ml) or carboxypeptidase A (1 U/ml) or 100 mMammoniumbicarbonate buffer of pH 8.0 containing dipeptidylaminopeptidase IV (0.5 U/ml). Experiments were initiated by addition ofan aliquot (100 l) of a stock solution (1 mg/ml) of the peptide in waterto 900 l preheated enzyme solution in an Eppendorf microvial giving aninitial concentration of 0.1 mg/ml (˜1.7·10-5-1.8·10-5 M) of thepeptide. The peptide/enzyme solution was kept at 37° C. and atappropriate time intervals samples of 100 l were withdrawn from thepeptide/enzyme solution and mixed thoroughly with 20 l 25% TFA inacetonitrile in order to stop the enzymatic degradation process. Theinactivated samples were transferred to autosampler vials and analysedfor content of intact test compound by HPLC as described below.Half-lives (t½) for the test compounds in enzyme solutions werecalculated from plots of natural logarithm to the residual concentration(i.e. HPLC peak heights) against time using the formula:

t _(1/2)=1/k _(obs)In(2), where k _(obs) is the apparent first-orderrate constant for the observed degradation.

HPLC Analysis

Samples from the stability experiments performed as described above wereanalysed by gradient HPLC analysis using the instrumentation describedabove and the following experimental conditions.

Column temperature: 30° C.

Injection volume: 10 l

Mobile phase A: 0.1% TFA in water

Mobile phase B: 0.085% TFA in acetonitrile (ACN)

Gradient: 32-52% B in 21 min

Detection: UV at 215 nm

The experimental results obtained from the individual stabilityexperiments are shown in Table 1 below. It appears from the table thatthe half life of the compounds of the invention is considerably extendedin solution with all enzymes tested.

TABLE 1 Test Compound Enzyme Solution Com- En- Half-life pound No. Namezyme Conc. (t_(1/2)) Com- H-(Gly⁸)-hGLP-1(7-36)- LAP 25 U/ml >3 dayspound 5 Lys(Palm)-Lys₆-NH₂ CPA 1 U/ml >2 days (SEQ ID NO: 153) DPP IV0.5 U/ml 440 min Com- H-(Gly⁸, Lys²⁶(Palm))- LAP 25 U/ml 1150 min pound7 hGLP-1(7-36)-Lys₆-NH₂ CPA 1 U/ml 1058 min (SEQ ID NO: 103) DPP IV 0.5U/ml 526 min Com- H-(Gly⁸, Lys³⁴(Palm))- LAP 25 U/ml ~1.5 day pound 6hGLP-1(7-36)-Lys₆-NH₂ CPA 1 U/ml >1 day (SEQ ID NO: 90) DPP IV 0.5 U/ml177 min GLP-1 H-hGLP-1(7-36)-NH₂ LAP 25 U/ml 152 min (SEQ ID NO: 114)CPA 1 U/ml 48 min DPP IV 0.5 U/ml 2.0 min Com- H-(Gly⁸)-hGLP-1(7-36)-LAP 25 U/ml ~1.5 day pound 4 Lys₆-NH₂ CPA 1 U/ml 145 min (SEQ ID NO: 88)DPP IV 0.5 U/ml 292 min Com- H-(Gly⁸)-hGLP-1(7-36)- LAP 25 U/ml 693 minpound (iii) NH₂ CPA 1 U/ml 127 min (SEQ ID NO: 87) DPP IV 0.5 U/ml 56min LAP: Leucine aminopeptidase, CPA: Carboxypeptidase A, DPP IV:Dipeptidyl aminopeptidase IV

23. In Vitro Stability Studies of Compound (iii) and Compound 4 in RatPlasma

The degradation of the two test compounds and in heparin stabilised rat(Sprague-Dawley) plasma was followed by the combination of solid phaseextraction and LC-MS. The degradation was followed for 720 minutes inplasma. The half-life of Compound (iii) was found to be 238 min. in ratplasma. This finding was compared with the half-life of Compound 4,which was found to be 466 min. in rat plasma.

Materials And Methods

Blank rat plasma in sodium heparin (5000 units/mL) were obtained fromHarlan Sera Lab Ltd. (Loughborough, UK). Test Substances and SolutionsThe test substances used in the study are listed in the table below. Forthe in vitro experiments a stock solution of 100 μg/ml milli-Q water wasused (corresponding to 26.0 μM Compound (iii) H-(Gly⁸)-1(7-36) (SEQ IDNO: 87) —NH₂ or 17.8 μM Compound 4).

Substance Name Batch No. Average Mw. Peptide Content Compound (iii) ZP7,73-1F 3284 g/mol 85% Compound 4 ZP 7,69-1C 4053 g/mol 72%

The LC-MS analysis was performed on an HP 1100 instrument consisting ofan on-line degasser, a quaternary gradient pump, an auto sampler, acolumn oven, Hewlett Packard (Wilmington, Del., USA) in combination witha Quattro Ultima mass spectrometer from Micromass (Altrincham, UK). Boththe LC and MS were controlled by MassLynx 3.3 software. The LCseparations prior to MS detection were performed on a Vydac 218MS52(2.1×250 mm) column (Hesperia, Calif., USA).

The initial plasma volume was 1000 μl (37° C.). From the initial plasmavolume, 100 μl was transferred to a 0.75 ml HPLC vial (used as blank),mixed with 560 μl extraction solution (MeCN:0.18 M ammonium carbonate pH9.5 (6:94 v/v), 4 C) and extracted by Solid Phase Extraction using ASPECXL4 Robot. A volume of 100 μl stock solution was added to the remaining900 μl plasma, mixed thoroughly and incubated at 37 C (corresponding toan initial concentration of 10 μg of the test compounds/ml). At eachtime point (0.2, 60, 120, 180, 240, 360, 480, 662 and 720 min.,respectively) 100 μl of the drug containing plasma was collected, mixedwith 560 μl ice cold extraction solution and immediately extracted bySPE as described above. The extracted plasma samples were analysed byLC-MS.

The LC-MS analysis were performed on an HP 1100 series LC in combinationwith a Quattro Ultima II triple quadrupole MS instrument.

The samples were kept at 18° C. in the autosampler tray prior toinjection of 10 μl. The separations were performed at 30° C. on a Vydac218MS52 (2.1×250 mm) LC column using a linear gradient from 15 to 50% Bwithin 14 min. at a flow rate of 250 μl/min. 0.1% formic acid in waterwas used as mobile phase A and 0.1% formic acid in MeCN as mobile phaseB. Compound 4 and Compound (iii) were detected by single ion recording(SIR) using the 6 H+ (m/z=676.7) and 4 H+ (m/z=822.1) ion species,respectively. The cone voltage for the analysis of compound (iii) andCompound 4 was set to 100 and 70 V, respectively.

The in vitro stability of Compound (iii) and Compound 4 have beeninvestigated in rat plasma by LC-MS. The degradation of the twocompounds were followed for 720 min. and the results were plotted as thenatural logarithm of the peak area vs. time. The degradation rates(k_(obs)) of the compounds were found as the slope after linearregression, and the half-life (T½) was found as ln 2/k_(obs). Theresults from the experiment are listed below.

Degradation Study over 720 minutes in Rat Plasma Compound T½ (min)k_(obs) (min⁻¹) r² Compound (iii) 238.4 0.0029 0.9785 Compound 4 466.10.0015 0.8596

The conclusion of the experiment is therefore that the provision of aC-terminal Lys₆ (SEQ ID NO: 9) peptide conjugation to the(Gly⁸)hGLP-1(7-36) (SEQ ID NO: 87) sequence results in a two foldincreased stability in rat plasma.

24. Single Dose Effect of Oral and Parenteral Administration of Compound5 on Blood Glucose Levels in Diabetic ob/ob Mice.

The compounds of the invention possess blood glucose loweringproperties. This was examined using Compound 5 to test the effect onblood glucose (BG) levels in the ob/ob mutant mice after intraperitoneal(i.p.) and peroral (p.o.) administration. Compound 5 reduced BG levelsin diabetic mice in a dose of 110 μg/mouse when administered i.p.Likewise p.o. administration of Compound 5 elicited a similar decreasein BG levels in a dose of 1100 μg/mouse, but not at lower doses.

Experimental

Forty female diabetic ob/ob mice (UmeA strain, Bomholtgaard), which areobese due to a dominant mutant leptin (Tomita, T., Doull, V., Pollock,H. G., and Krizsan, D. 1992. Pancreatic islets of obese hyperglycemicmice (ob/ob). Pancreas 7: 367-75) were housed (3 mice/cage) undercontrolled ambient conditions following a 12:12-h light:dark cycle andfed standard Altromin no 1324 diet with free access to tap water. Atarrival the animals were 8 weeks of age. The mice were allowed 2 weeksof acclimatization before experiments were initiated. At the time ofexperiment the mice were 13 weeks old with a body weight of 41.8±3.2 g(mean±SD; n=42). Handling of the mice one and three days before theexperiment was performed in order to reduce stress-induced BGexcursions. On the day of the experiment, blood was taken from the tipof the tail 2-3 hours after the light was turned on. A single drop ofblood (<5 μl) was dropped on the glucose strip for analysis and measuredby an Elite Autoanalyser, Bayer, Denmark. Whole blood glucose (BG)concentration was analysed by the immobilised glucose oxidase method.Blood glucose levels varied between normoglycaemia and severehyperglycaemia (range: 3.6-15.6 mM; mean±SD: 9.4±3.3 mM; n=42). Sixanimals with BG<5.8 mM were excluded from the study (total n=36). Theremaining animals were stratified based on their BG levels in order toensure that the mean BG was similar among groups. One hour after theinitial control blood sampling, drugs were administered and BG wasmeasured at t=60 min, t=120 min, t=240 min, t=480 min.

Peptides and Other Materials

Compound 5 (batch nr. ZP 3.12 fraction 1-2. Purification) wassynthesised by the Department of Chemistry, Zealand Pharmaceuticals. Thepeptide was dissolved in sterile isotonic NaCl shortly before dosing andgiven in a volume of 0.2 ml. The same solutions were used for both p.o.and i.p. administration. For each animal, a data log sheet was filledout at the time of each blood sampling.

Drug Administration

Animals were administered with Compound 5, and the maximum dose was 1100μg/mouse and the lowest dose was 1.1 μg/mouse. As a negative control,saline was administered p.o. and as positive control the test compoundwas given i.p. in a dose of 110 μg/mouse.

During control conditions, BG levels in non-fasted ob/ob mice weresimilar in all groups (individual group data not shown), but withingroups, there was a great scatter on BG levels (BG range for allanimals: 5.8-15.6 mM). Therefore, to correct for the varying degree ofhyperglycemia, results are expressed as the relative difference frombaseline (% control). Intraperitoneal administration of 110 μg Compound5 produced a sustained decrease in BG that reached nadir at 1-2 hrsafter administration of the compound. No changes were observed in salinetreated animals. In most groups (5/6), BC increased between 4 and 8 hrsafter drug administration. Compound 5 reduced the BG levels in a dose of110 μg/mouse when administered i.p. in diabetic ob/ob mice (data notshown). The antidiabetic effect was observed after 60 minutes and wasmaximal 2-4 h after administration of the compound. Furthermore, along-lasting effect (>8 hours) suggests that Compound 5 has a longerduration of action than the notoriously short-acting native GLP-1(Bailey, C. J. & Flatt, P. R. 1987. Glucagon-like peptide-1 and theentero-insular axis in obese hyperglycaemic (ob/ob) mice. Life Sci, 40,521-5). The dose 1100 μg/mouse p.o. elicited a similar decrease in BG asobserved in animals treated with 110 μg i.p.

We have shown that Compound 5 effectively lowers BG levels in diabeticob/ob mice following i.p. administration of 110 μg/mouse the compound. Asimilar effect is seen after 1100 μg/mouse of Compound 5 when given bythe oral route. This suggests that the compound is absorbed from thegastrointestinal tract.

25. In Vivo Studies With

Compound 1 (des Pro³⁶-exendin-4(1-39)-NH₂ (SEQ ID NO:101)),

Compound 2 (des Pro³⁶-exendin-4(1-39)-Lys6-NH₂ (SEQ ID NO:93)),

Compound (iii) (Gly⁸-GLP1-(7-36)(Human)-NH₂ (SEQ ID NO:87)),

Compound 4 (Gly⁸-GLP1-(7-36)(Human)-Lys₆-NH₂ (SEQ ID NO:88)) and

Compound 5 (Gly⁸Lys³⁷(palmitoyl)-GLP1-(7-36)(Human)-Lys₇-NH₂ (SEQ IDNO:89))

Various concentrations of each peptide are administered orally andintraperitoneally to ob/ob mice to determine if these compounds affectblood glucose levels. The experimental conditions used were the same asdescribed in Example 24.

Peptides and Other Materials

Des Pro³⁶-exendin-4(1-39)-NH₂ (Compound 1, SEQ ID NO:101) and the samepeptide, but with an additional sequence, Lys₆, attached at theC-terminal, des Pro³⁶-exendin-4(1-39)-Lys6-NH₂ (Compound 2, SEQ IDNO:93), Gly⁸-GLP1-(7-36)(Human)-NH₂ (Compound (ii), SEQ ID NO:87) andthe same peptide, but with an additional sequence, Lys₆, attached at theC-terminal, Gly⁸-GLP1-(7-36)(Human)-Lys₆-NH₂ (Compound 4, SEQ ID NO:88)and Gly⁸Lys³⁷(palmitoyl)-GLP1-(7-36)(Human)-Lys₇-NH₂ (Compound 5, SEQ IDNO:89) are synthesized using methods described above. Solutions areprepared on the morning of dosing, immediately before the animals areadministered. The same solutions are used for both peroral andinterperitoneal administration. All peptides are dissolved in sterileisotonic NaCl and given in a volume of 0.2 ml. All experiments arecarried out in the same mice to compare the active doses of the peptidesshown in Table 2. Blood sampling is performed as described above and theanimals are administered with the doses shown in Table 3. As negativecontrol, saline is administered perorally. Results are shown in Table 4.

TABLE 2 Number Compound Compound 1 des Pro³⁶-exendin-4(1-39)-NH₂ (SEQ IDNO: 101) Compound 2 des Pro³⁶-exendin-4(1-39)-Lys₆-NH2 (SEQ ID NO: 93)Compound (ii) Gly⁸-GLP1-(7-36)(Human)-NH2 (SEQ ID NO: 87) Compound 4Gly⁸-GLP1-(7-36)(Human)- Lys₆-NH2 (SEQ ID NO: 88) Compound 5Gly⁸Lys³⁷(palmitoyl)-GLP1-(7-36)(Human)- Lys₇-NH₂ (SEQ ID NO: 89)

TABLE 3 Group 5 Dose Group 1 Group 2 Group 3 Group 4 peroral Group 6Dose Dose Dose Dose μl/mouse Dose peroral peroral peroral peroralIsotonic i.p Compound μg/mouse μg/mouse μg/mouse μg/mouse salineμg/mouse Compound 1 400 40 4 0.4 200 μl 40 Compound 2 1000 100 10 1 200μl 100 Compound (ii) 1000 100 10 1 200 μl 100 Compound 4 1000 100 10 1200 μl 100 Compound 5 1000 100 10 1 200 μl 100

Group data were summarised as the mean±SEM of the individual results ineach treatment group. In order to analyse the effects of the compounds,the absolute and the relative (% of t=0) difference from baseline wascalculated for each time point.

TABLE 4 0 1 hour 2 hours 4 hours Compound 1-saline 100 103 107 92Compound 1-400 μg po 100 93 88 93 Compound 1-40 μg po 100 89 89 91Compound 1-4 μg po 100 105 88 91 Compound 1-0.4 μg po 100 106 103 100Compound 1-40 μg ip 100 68 69 74 Compound 2-saline 100 100 112 114Compound 2-1000 μg po 100 67 69 64 Compound 2 100 μg po 100 78 71 72Compound 2 10 μg po 100 86 72 72 Compound 2 1 μg po 100 112 101 96Compound 2 100 μg ip 100 75 67 63 Compound (ii) -saline 100 95 87 100Compound (ii) -1000 μg po 100 87 105 94 Compound (ii) -100 μg po 100 118111 92 Compound (ii) -10 μg po 100 101 94 104 Compound (ii) -1 μg po 10094 89 96 Compound (ii) -100 μg ip 100 70 60 81 Compound 4 -saline 100102 94 79 Compound 4 -1000 μg po 100 128 72 78 Compound 4 -100 μg po 10072 70 58 Compound 4 -10 μg po 100 98 95 81 Compound 4 -1 μg po 100 99 8984 Compound 4 100 μg ip 100 83 58 56 Compound 5 -saline 100 90 86 103Compound 5 -1000 μg po 100 73 75 67 Compound 5 -100 μg po 100 97 140 107Compound 5 -10 μg po 100 90 120 126 Compound 5 - 1 μg po 100 111 133 114Compound 5 - 100 μg ip 100 63 50 52

The results obtained are shown in Table 4 and in FIGS. 1-3.

These results show that all tested compounds have an effect in loweringblood glucose levels. The effect is most pronounced when Compound 1 isgiven intraperitoneally whereas the effect of 1000 μg po of Compound 2is comparable to the effect of 100 μg ip of Compound 2. The potency ofCompound 1 (des Pro³⁶-exendin-4(1-39)-NH₂, SEQ ID NO:101) and Compound 2(des Pro³⁶-exendin-4(1-39)-Lys₆-NH₂, SEQ ID NO:93) when givenintraperitoneally is shown to be very similar to exendin-4(1-39)-NH₂(SEQ ID NO: 102) (Compound (i)) itself (data not given) administered inthe same way.

For Compound 1, des Pro³⁶-exendin-4(1-39)-NH₂ (SEQ ID NO:101), there isno effect in lowering blood glucose levels up to a dose of 400 μg/mousewhen the compound is administered perorally, whereas for the samecompound with the addition of the Lys6 fragment there is activity seenat a dose of 10 μg/mouse. This indicates that the minimum effective oraldose of the des Pro³⁶-exendin-4(1-39)-Lys₆-NH2 (SEQ ID NO:93) is atleast 40 times lower than for des Pro³⁶-exendin-4(1-39)-NH₂ (SEQ IDNO:101).

These results show that the attachment of the sequence Z has nosignificant effect on the potency of the various peptides whenadministered interperitoneally while significantly enhancing the potencyof the compound when administered perorally.

26. Bioavailability of Compound 4 and Compound (iii) AfterGastro-Intestinal Delivery in Duodenum in Conscious Rats.

Various peptide based GLP-1 analogues have been developed for parenteraluse, but none of these substances has been pharmacologically effectiveafter oral administration [Hoist, J. J.: Enteroglucagon. Annu RevPhysiol, 59:257-271, 1997]. It was decided to examine the absorption ofthe test compound from the duodenum in conscious rats. Compound (iii)(Gly⁸)hGLP-1(7-36)-NH₂ (SEQ ID NO: 87) was used as reference.

Chemicals and Reagents

Blank rat plasma in sodium heparin (5000 units/mL) were obtained fromHarlan Sera Lab Ltd. (Loughborough, UK). OASIS™ HLB solid phaseextraction columns, 1 cc, 30 mg sorbent, were obtained from Waters(Milford, Mass., USA) and ISOLUTE C18 (EC), 1 cc, SPE columns wereobtained from IST (Mid Glamorgan, U.K.). The LC/MS analysis wasperformed on a HP 1100 instrument consisting of an on-line degasser, abinary gradient pump, an auto sampler, a column oven, Hewlett Packard(Wilmington, Del., USA) in combination with a Quattro Ultima massspectrometer from Micromass (Altrincham, UK) both the LC and MS werecontrolled by MassLynx 3.3 software. The LC separations prior to MSdetection were performed on a Vydac 218MS52 (2.1×250 mm) column(Hesperia, Calif., USA).

Drugs and Dose Levels:

Compound 4 (batch No. ZP 7.97-5-F, 4053 g/mol) and Compound (iii) (batchNo. ZP 7.73-2-G, 3854 g/mol) were synthesised in-house using the Fmocstrategy. The identification was performed by mass spectrometry and thepurity of both batches was determined by RP-HPLC to 97 and 99.7% for thetest compounds, respectively. The peptide content of the batches were72% and 80% for ZP 7.97-5-F and ZP 7.73-2-G, respectively. The peptideswere dissolved in pyrogen free isotonic saline and doses of 1.000 or10.000 nmol/kg administered through the intra duodenal catheter in avolume of 100 μl.

Animals:

Fourteen Sprague-Dawley rats weighing 250 to 350 g. were used for theexperiment. The rats were anaesthetised with Hypnorm®-Dormicum® s.c. anda catheter was inserted into the femoral artery for arterial bloodsampling. An additional catheter was inserted into the duodenum via anincision in the ventricle. Before the experiment was started, the ratswere allowed to recover for one week after the operation. The operatedrats were conscious at the day of the experiment. In order to establishwhether the intra duodenal catheters were situated in the duodenum, anautopsy was performed on the rats immediately after the experiment.

Sample Treatment:

Blood samples were collected at t=−5, 5, 10, 15, 20, 40, and 60 min. Theblood was collected in EDTA containing ice-chilled tubes and immediatelycentrifuged at 4° C. for 5 min (4.000×g). Plasma (250 μl) wastransferred to ice-chilled 0.75 ml PLC vials containing 250 μlextraction solution (MeCN: 0.18 M Ammonium Carbonate pH 9.5, 10:90 v/v).The plasma samples were stored at −20? C until SPE and LC/MS analysis.

Solid Phase Extraction:

The drug containing plasma samples (400 μl) were loaded onto solid phaseextraction columns preconditioned with 950 μl MeCN followed by 950 μlwater. The columns were washed with 950 μl 2% TFA in water followed byan equal volume of 2% TFA in MeCN:water (20:78 v/v). The analytes wereeluted with 500 μl 2% TFA in MeCN:water (60:38 v/v) and analysed byLC/MS.

LC/MS

The samples were kept at 18° C. in the auto sampler tray prior toinjection of 20 to 50 μl onto the LC column (Vydac 218MS52 (2.1×250 mm).The separations were performed at 30° C. using a flow rate of 250 μl/minand a gradient according to Table 1. Both the test compound and thereference drug were detected by single ion recording (SIR) using them/z=676.7 and the m/z=1095.2 and 821.8 ion species, respectively. Allinstrument conditions were controlled by MassLynx software ver. 3.3software.

Compound Gradient Compound 4 Initial: 15% B, 0-14 min; 15-50% B, 14-15min; 50-15% B and 15-20 min 15% B. Compound (iii) Initial: 25% B, 1-1.5min; 25-30% B, 1.5-10 min; 30-40% B, 10-10.5 min; 40-90% B, 11.5-12 min;90-25% B, and 12-17 min 25% B. The gradient used for the analysis of thetest compounds using 0.1% formic acid in water or MeCN as Mobile phase Aor B, respectively.

The plasma samples were analysed as described under materials andmethods. The bioavailability of Compound 4 was examined in doses of1.000 (n=4) and 10.000 (n=5) nmol/k, whereas Compound (iii) was onlystudied in a dose of 10.000 (n=5) nmol/kg. At all the investigated timepoints the concentration of Compound (iii) was below the detection limit(approx. 0.5 nM), the exact bioavailability could therefore not beestimated. In contrast, Compound 4 was detected in the plasma samplesfrom two out of four rats after intra duodenal administration of 1.000nmol/kg and in four out of five rats following administration of 10.000nmol/kg.

27. In Vivo Pharmacokinetics of Compound 1, Compound 2, Compound 4, andCompound (iii) After I.V. Administration to Rabbits and Pigs

We have shown an increased in vitro stability of the GLP-1 agonistCompound 4 when compared to the reference drug Compound (iii) in ratplasma. In order to establish whether this effect is sustained in vivo,the pharmacokinetic parameters of the two compounds are examined inrabbits. Using the same experimental conditions these parameters werealso measured for Compounds 1 and 2 in rabbits and using similarconditions in pigs.

Chemicals and Reagents

Blank rabbit plasma in sodium heparin (5000 units/mL) were obtained fromHarlan Sera Lab Ltd. (Loughborough, UK). OASIS™ HLB solid phaseextraction columns, 1 cc, 30 mg sorbent, were obtained from Waters(Milford, Mass., USA) and ISOLUTE C18 (EC), 1 cc, SPE columns wereobtained from IST (Mid Glamorgan, U.K.). The LC/MS analysis wasperformed on a HP 1100 instrument consisting of an on-line degasser, abinary gradient pump, an auto sampler, a column oven, Hewlett Packard(Wilmington, Del., USA) in combination with a Quattro Ultima massspectrometer from Micromass (Altrincham, UK) both the LC and MS werecontrolled by MassLynx 3.3 software. The LC separations prior to MSdetection were performed on a Vydac 218MS52 (2.1×250 mm) column(Hesperia, Calif., USA).

Drugs and Dose Levels:

Compound 4 (batch No. ZP 7.97-5-F, 4053 g/mol) and Compound (iii) (batchNo. ZP 7.73-2-G, 3854 g/mol) were synthesised in-house using the Fmocstrategy. The identification was performed by mass spectrometry and thepurity of both batches were determined by RP-HPLC to 97 and 99.7% forthe test compounds, respectively. The peptide content of the batcheswere 72% and 80% for ZP 7.97-5-F and ZP 7.73-2-G, respectively. Thepeptides were dissolved in pyrogen free isotonic saline and bothpeptides were administered i.v. to rabbits and rats using a dose of 1000nmol/kg.

Rabbits:

Fifteen New Zealand White rabbits weighing 2.5 to 3.0 kg were used forthe experiment. On the day of the experiment, the rabbits wereanaesthetised with Hypnorm® i.m. followed by Dormicum® i.v. Catheterswere inserted into the femoral vein and artery for i.v. administrationof drugs and arterial blood sampling. The rabbits stayed unconsciousthroughout the experiment.

Sample Treatment:

Blood samples were collected at t=1, 3, 5, 10, 15, 20, 30, 40, 60, 90,120, 150, 180, and 240 min. The blood was collected in EDTA containingice-chilled tubes and immediately centrifuged at 4 C for 5 min(20.000×g). Plasma (250 μl) was transferred to ice-chilled 0.75 ml PLCvials containing 250 μl extraction solution (MeCN: 0.18 M AmmoniumCarbonate pH 9.5, 10:90 v/v). The plasma samples were stored at −20 Cuntil SPE and LC/MS analysis.

Solid Phase Extraction:

The drug containing plasma samples (400 μl) are loaded onto OASIS™ HLB(Compound 4) or ISOLUTE™ (Compound (iii)) solid phase extraction columnspreconditioned with 950 μl MeCN followed by 950 μl water. The columnsare washed with 950 μl 2% TFA in water followed by an equal volume of 2%TFA in MeCN:water (20:78 v/v). The analytes are eluted with 500 μl 2%TFA in MeCN:water (60:38 v/v) and analysed by LC/MS.

LC/MS

The samples were kept at 18 C in the auto sampler tray prior toinjection of 20 to 50 μl onto the LC column (Vydac 218MS52 (2.1×250 mm).The separations were performed at 30° C. using a flow rate of 250 μl/minand a gradient according to the table below. Both the test compound andthe reference drug are detected by single ion recording (SIR) using them/z=676.7 and the m/z=1095.2 and 821.8 ion species, respectively. Allinstrument conditions were controlled by MassLynx software ver. 3.3software.

Compound Gradient Compound 4 Initial: 15% B, 0-14 min; 15-50% B, 14-15min; 50-15% B and 15-20 min 15% B. Compound (iii) Initial: 25% B, 1-1.5min; 25-30% B, 1.5-10 min; 30-40% B, 10-10.5 min; 40-90% B, 11.5-12 min;90-25% B, and 12-17 min 25% B. The gradient used for the analysis of thetest compounds using 0.1% formic acid in water or MeCN as Mobile phase Aor B, respectively.

The plasma samples were analysed as described under materials andmethods and the plasma concentration (C_(pl)) plotted versus time in asemi log diagram. The plasma concentration were followed for three hoursin rabbits, whereas the limited blood volume of rats restricted theblood sampling in this specie to one hour. The C_(pl) vs. time curvesfrom the individual rabbits were fitted to a two-compartment open model(figure not shown) using 1/y² weighted least squares in WinNonlin 3.1(Pharsight Corp. (Mountain View, Calif.)). The pharmacokinetic constantsobtained from the data analysis are listed in Table 5 and thedegradation kinetics in rabbit after i.v. injection of 1 μmol/kg ofCompound 4 and Compound (iii), respectively, is shown in FIG. 4.

TABLE 5 In vivo kinetics in rabbits and pigs ** Comp. (iii) Comp. 4Comp. 1 Comp. 2 Comp. 2 ** Param- (n = 7) (n = 8) (n = 5) (n = 5) (n =2) eter Mean Mean Mean Mean Mean T_(1/2), 2.3 6.8 4.4 11 16 α minT_(1/2), 10.8 28.0 23 69 252 β min

Table 5: The pharmacokinetic constants were obtained from rabbits whenthe C_(pl) vs. time curves was fitted mathematically. The compounds wereadministered iv in a concentration of 1000 nmol/kg. T_(1/2) values aregiven in minutes (min) for the α and β phase. Statistics: two-tailedt-test assuming samples with unequal variances showed p<0.001 for allmeasured parameters. In conclusion the T1/2 value for Compound 4 isapproximately three times the value for the reference Compound (iii)and, likewise, the T1/2 value for Compound 2 is approximately threetimes the value calculated for Compound 1 which represents theunconjugated equivalent.

28.Glucose Tolerance Test of Compounds 2 14-16 18 and 19 Compared toCompound (i)

Male diabetic db/db mice (M&B, Bomholdtgaard, LI. Skensved, Denmark) areused. This well-described mouse model has inherited malfunctions of theglucose metabolism due to a mutation in the leptin receptor. Like humanpatients with uncontrolled non-insulin demanding diabetes mellitus(NIDDM), homozygous db/db mice experience polydipsia, polyuria andglycosuria and gain weight during their first 3 months of life despitetheir hyperglycaemic stage. However, in this model the hyperglycaemia isassociated with progressive pancreatic islet atrophy with possibleketosis and death at 6-8 months of age. Thus, attention should be paidto the progression and status of their disease state. Therefore,preferably only db/db mice less than 16 weeks old should be used fordrug testing og GLP-1 analogues.

All animals are acclimatised for at least one week and handled daily fortwo days prior to the first oral glucose tolerance test (OGTT).Furthermore, to reduce stress-induced glucose excursions, the animalsshould be subjected to at least one OGTT without compound as describedbelow prior to the experiment. Due to the great scatter of glucosetolerance among diabetic mice, the animals are stratified by an OGTTprior to their first use.

Peptides

Peptides are dissolved in 0.1 M phosphate-buffered saline (PBS) with0.1% bovine albumin where pH is adjusted to 7.4 by adding 5 M NaOH. Allsolutions are prepared fresh on the morning immediately before theexperiment. Vehicle treated animals are given PBS with 0.1% albuminalone.

Glucose Tolerance Test and Dosing

Before the oral glucose tolerance test, the animals are fasted for 17hours (from 4 p.m. until 9 a.m. the following morning). Beginning at9.00 a.m. blood is taken from the tail tip (t=−15 min) and blood glucoseis measured. The whole blood glucose (mM) concentration is analysed bythe immobilised glucose oxidase method using a drop of blood (<5 μl,Elite Autoanalyser, Bayer, Denmark) following the manufacturer's manual.Animals with severe diabetes (>10 mM) are excluded. Immediately afterthe initial blood sample, the animals receive an intraperitoneal (i.p.)injection of vehicle or a dose of antidiabetic compound. Injectionvolume is 200 μl/50 g body weight in all groups. Fifteen minutes afteri.p. administration of the substance an oral dose of 1 g/kg glucose(Sigma, St. Louis) dissolved in water (200 μl/50 g body weight) isgiven, and the animals are returned to their home cages (t=0). Bloodglucose levels are measured at t=30 min, t=60 min, t=120 min and t=240min. The animals are fasted during the observation period. For eachanimal a data log sheet was filled in at the time of each bloodsampling.

Calculations and Statistics

In order to analyse the effects of the compounds, the absolute and therelative difference from baseline (t=0) are calculated for each timepoint. The area under the curve for the whole experiment (AUC 0-240 min)is determined using the trapezoid method. On the day of stratification,the mice are distributed in order to ensure that the glucose tolerancesare similar in all groups. However, to correct for the progression ofthe diabetes with time, a vehicle treated control group is tested oneach day of experiment and the response to drugs are expressed relativeto response observed in vehicle-treated time-control animals.Dose-response curves for each substance are plotted, cf. FIG. 5, and theeffect of drug relative to responses obtained during treatment withvehicle are analysed using an ANCOVA analysis (analysis of covariance).Treatment (drug or vehicle) is considered the independent variable, AUC0-240 min expressed as per cent response in vehicle-treated time-controlmice is the dependent variable, and drug dose is defined as covariate.Post-hoc analysis is performed using Fisher's Least Significant test.Differences are considered significant at the 0.05 level. Statisticalanalyses were performed using Statistica version 5.5 for Windows NT,StatSoft, Tulsa, Okla., U.S.A. The dose response curves shown in FIG. 5clearly shows that all tested compounds exhibit a glucose loweringeffect comparable to that of the reference drug.

29. Effects of Compound 2 and Compound (i) on OGGT in db/db Mice

FIG. 7 is a plot of AUC for Compound 2 and Compound (i) in an OGTTperformed using the same experimental conditions as described in Example28. The figure shows that the blood glucose lowering effect of Compound2 is the same as the effect of the prior art compound (iii).

30. Long Term Effects of Compound 2, 100 nmol/kg i.p. on the OralGlucose Tolerance Test (OGTT) when Administered up to 24 Hours Beforethe OGTT

This experiment uses the maximal dose of 100 nmol/kg i.p. in db/db miceand otherwise, the same experimental conditions as described in Example28 are used. Results are shown in FIG. 8 and the conclusion of theexperiment is that the duration of action of Compound 2 is up to 18hours in db/db mice.

The invention described and claimed herein is not to be limited in scopeby the specific embodiments herein disclosed, since these embodimentsare intended as illustrations of several aspects of the invention. Anyequivalent embodiments are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims. Variousreferences are cited herein, the disclosure of which are incorporated byreference in their entireties.

1. (canceled)
 2. A method for treating type 2 diabetes mellitus in asubject in need thereof, said method comprising administering to saidsubject (i) a peptide conjugate comprising an exendin-4 variant coupledvia its C-terminus to an amino acid sequence Z, wherein said exendin-4variant comprises a sequence characterized by deletion of between 1 and5 amino acid residues at positions corresponding to positions 34-38 ofexendin-4(1-39) and wherein Z comprises from 4 to 20 Lys amino acidresidues, or a pharmaceutically acceptable salt thereof; and (ii) anantidiabetic agent each in an amount that together is effective for thetreatment of type 2 diabetes mellitus.
 3. The method of claim 2, whereinsaid antidiabetic agent is insulin or an oral hypoglycaemic agent. 4.The method of claim 3, wherein said antidiabetic agent is an oralhypoglycaemic agent selected from metformin, a sulfonyl urea, or athiazolidinedione.
 5. The method of claim 2, wherein said peptideconjugate is administered parenterally.
 6. The method of claim 5,wherein said peptide conjugate is administered subcutaneously.
 7. Themethod of claim 5, wherein said peptide conjugate is parenterallyadministered in a liquid dosage form.
 8. The method of claim 2, whereinsaid peptide conjugate is formulated as a lyophilized solid andreconstituted prior to administration to said subject.
 9. The method ofclaim 2, wherein said exendin-4 variant comprises a sequencecharacterized by deletion of between 1 and 3 amino acid residues atpositions corresponding to positions 34-38 of exendin 4(1-39).
 10. Themethod of claim 9, wherein said exendin-4 variant comprises a sequencecharacterized by deletion of 1, 2, or 3 Pro residues at positionscorresponding to positions 36-38 of exendin-4(1-3 9).
 11. The method ofclaim 2, wherein said amino acid sequence Z is Lys₄, Lys₅, Lys₆, Lys₇,Lys₈, Lys₉, or Lys₁₀.
 12. The method of claim 2, wherein said peptideconjugate is administered as a free acid.
 13. The method of claim 2,wherein said peptide conjugate is administered as a pharmaceuticallyacceptable salt.
 14. The method of claim 2, wherein said exendin-4variant is selected from desPro³⁶ exendin-4 (1-39),desPro³⁶Pro³⁷-exendin-4(1-39), and desPro³⁶Pro³⁷Pro³⁸-exendin-4(1-39).15. The method of claim 14, wherein said peptide conjugate is selectedfrom desPro³⁶-exendin-4(1 -3 9)-Lys₆-NH₂,desPro³⁶Pro³⁷-exendin-4(1-39)-Lys₆-NH₂,desPro³⁶Pro³⁷Pro³⁸-exendin-4(1-39)-Lys₆-NH₂, and pharmaceuticallyacceptable salts thereof.
 16. The method of claim 15, wherein saidpeptide conjugate is selected from desPro³⁶-exendin-4(1-39)-Lys₆-NH₂,and pharmaceutically acceptable salts thereof.
 17. The method of claim16, wherein said antidiabetic agent is selected from insulin, metformin,a sulfonyl urea, and a thiazolidinedione.
 18. The method of claim 16,wherein said antidiabetic agent is insulin.